Labyrinthulid microorganism capable of producing microbial oil, microbial oil, methods for producing said microorganism and for producing said microbial oil, and uses of said microorganism and said microbial oil

ABSTRACT

A Labyrinthulid microorganism capable of producing a PUFA only through the elongase-desaturase pathway. A Labyrinthulid microorganism which has no ability of producing a PUFA through the endogenous PUFA-PKS pathway or has the ability at an extremely weak level and which has an ability of producing a PUFA through the endogenous elongase-desaturase pathway. The Labyrinthulid microorganism is a microorganism belonging to the genus Parietichytrium or Parietichytrium.

TECHNICAL FIELD

The present invention relates to microbial oil obtained from alabyrinthulid, a labyrinthulid capable of producing microbial oil, amethod for producing the microbial oil and the labyrinthulid, and a useof the microbial oil and the labyrinthulid.

More specifically, the present invention relates to polyunsaturatedfatty acids (PUFAs) and the production thereof, and relates to alabyrinthulid genetically modified such that the fatty acid compositionis modified, preferably a labyrinthulid that produces PUFAs only via theelongase-desaturase pathway, a method for producing PUFAs using thesame, lipids (microbial oils) containing PUFAs produced using the same,and uses thereof.

BACKGROUND ART

Labyrinthulids are eukaryotic microorganisms belonging to the classLabyrinthulomycetes, which includes the order Labyrinthulales and theorder Thraustochytriales are known to be universally present in oceans.

Microorganisms belonging to the order Thraustochytriales are alsogenerically called thraustochytrids.

Labyrinthulids have drawn attention as industrial oil-producingmicroorganisms. DHA produced by labyrinthulids has been productized asDHA-containing lipid raw materials, high DHA-containing animal feeds,and the like (Non-patent Document 1). Specific examples include breedingtechniques for the genus Thraustochytrium and genus Schizochytrium(Patent Document 1), and techniques for using thraustochytrids and ω-3HUFAs (highly unsaturated fatty acids) extracted from thraustochytrids(Patent Document 2).

It is generally known that PUFAs are biosynthesized via theelongase-desaturase pathway (also called the standard pathway), but ithas also been shown that certain labyrinthulids produce PUFAs via adifferent pathway, namely a metabolic pathway using polyketide synthase(PKS) (Non-patent Document 2). In the present invention, this pathwaywill hereinafter be called the PUFA-PKS pathway or the PKS pathway. Thecompositions of PUFAs produced via this pathway are mostly DHA and n-6DPA.

There are some scientists who think that labyrinthulids (particularlythraustochytrids) have only the PUFA-PKS pathway as a PUFA biosynthesispathway, and do not have the general elongase-desaturase pathway foundin other organisms (Non-patent Document 3). Actually, it has beenreported that by gene-isruption of the PUFA-PKS pathway, somelabyrinthulids are rendered lethal and such labyrinthulids cannot begrown unless PUFAs are added to the culture medium (Non-patent Document3). This result means that since PUFAs are required for growth of theselabyrinthulids and the gene of the PUFA-PKS pathway, which is the solePUFA biosynthesis pathway, has been disrupted, the nature of thelabyrinthulids has changed into exogenous PUFAs requirement for growth.

However, contrary to common knowledge of persons skilled in the art,some labyrinthulids having both the PUFA-PKS pathway and theelongase-desaturase pathway as PUFA biosynthesis pathways have beenfound through our examinations. These are specifically described in thespecification of Patent Document 3 and in Non-patent Document 5.

Citing Thraustochytrium aureum ATCC 34304 as an example, it has beenfound that this strain has a Δ12 desaturase gene, which is an entryenzyme of the elongase-desaturase pathway. Furthermore, it has beenfound that a strain in which this gene was disrupted by homologousrecombination accumulates predominantly oleic acid, which is thesubstrate of Δ12 desaturase, compared to the wild-type strain, and thatdecreases in both linoleic acid of the product and PUFAs locateddownstream of the biosynthesis system thereof were observed.Additionally, it has been found that because this strain is capable ofproducing PUFAs via the elongase-desaturase pathway, by disruption ofthe PUFA-PKS pathway gene it is not rendered lethal. This report was thefirst to show that the elongase-desaturase pathway also functions as aPUFA biosynthesis pathway in labyrinthulids, and was discussed inNon-patent Document 6 as well.

At the start, Non-patent Document 6 states that labyrinthulids thatproduce DHA as the main fatty acid are widely used industrially. Incontrast, the creation of labyrinthulids containing desired PUFAs otherthan DHA as the main fatty acid was first enabled by the discovery oflabyrinthulids having both the PUFA-PKS pathway and theelongase-desaturase pathway and by application of transformationtechniques to these labyrinthulids (Patent Document 3, Non-patentDocument 7). That is, it is possible to create strains containingcertain PUFAs other than DHA as the main fatty acid by first disruptingthe genes of the PUFA-PKS pathway with homologous recombinationtechnique, and then disrupting or overexpressing the genes of theenzymes constituting the elongase-desaturase pathway appropriately.Example 12 of Patent Document 3 is cited as a specific example. In thisexample, the PUFA-PKS pathway genes of Thraustochytrium aureum ATCC34304 were disrupted, and then the C20 elongase gene was disrupted. Anω3 desaturase gene derived from Saprolegnia diclina was thentransformed, thereby successfully creating a strain in which arachidonicacid increased approximately 6-fold. EPA increased approximately10-fold, and DHA decreased approximately 1/16 in comparison with thewild-type strain.

CITATION LIST Patent Documents

-   Patent Document 1: JP 3127161 B-   Patent Document 2: JP 3669372 B-   Patent Document 3: WO 2012/043826-   Patent Document 4: US 2005/0,014,231 A

Non-Patent Documents

-   Non-patent Document 1: Zvi Cohen et al. editors, “Single Cell Oils    Microbial and Algal Oils 2nd edition”, (U.S.), AOCS Press, 2010, p.    88-   Non-patent Document 2: Metz J G, Roessler P, Faccioti D, et al.    Production of polyunsaturated fatty acids by polyketide synthesis in    both prokaryotes and eukaryotes. Science 2001; 293: 290-293-   Non-patent Document 3: Ratledge C. Omega-3 biotechnology: Errors and    omissions, Biotechnology Advances 30 (2012) 1746-1747-   Non-patent Document 4: Lippmeier J. C. et al., Lipids, 44 (7),    621-630 (2009)-   Non-patent Document 5: Matsuda T., Sakaguchi K, Hamaguchi R,    Kobayashi T, Abe E, Hama Y, Hayashi M, Honda D, Okita Y, Sugimoto S,    Okino N, Ito M. The analysis of delta12 fatty acid desaturase    function revealed that two distinct pathways are active for the    synthesis of polyunsaturated fatty acids in Thraustochytrium aureum    ATCC 34304. J. Lipid Res. 53 (6): 1210-1222 (2012)-   Non-patent Document 6: ASBMB Today, June 2012, p. 30-   Non-patent Document 7: Sakaguchi K, et al., Versatile Transformation    System That Is Applicable to both Multiple Transgene Expression and    Gene Targeting for Thraustochytrids. Appl. Environ. Microbiol. 78    (9): 3193-3202 (2012)-   Non-patent Document 8: Yazawa K., Lipids. 31, Supple. 297-300 (1996)-   Non-patent Document 9: Journal of the Japan Society for Bioscience,    Biotechnology and Agrochem, 77, 2, 150-153 (2003)-   Non-patent Document 10: “Illustrated Bio Experiments Vol. 2    Fundamentals of Gene Analysis”, p. 63-68, Shujunsha, published 1995-   Non-patent Document 11: Sanger, F. et al., Proc. Natl. Acad. Sci.,    74, 5463 (1997)-   Non-patent Document 12: Cigan and Donahue, 1987; Romanos et al.,    1992-   Non-patent Document 13: Ausubel F. M. et al., Current Protocols in    Molecular Biology, Unit 13 (1994)-   Non-patent Document 14: Guthrie C., Fink G. et al., Methods in    Enzymology: Guide to Yeast Genetics and Molecular Biology, Volume    194 (1991)-   Non-patent Document 15: Qiu, X. et al. J. Biol. Chem., 276, 31561-6    (2001)-   Non-patent Document 16: Abe E., et al., J. Biochem., 140, 247-253    (2006)-   Non-patent Document 17: “Illustrated Bio Experiments Vol. 2    Fundamentals of Gene Analysis”, p. 117-128, Shujunsha, published    1995-   Non-patent Document 18: DIG Manual (Japanese Edition) 8th, Roche    Applied Science

SUMMARY OF THE INVENTION Problem that the Invention is to Solve

The present invention is directed to provide a labyrinthulid geneticallymodified such that the fatty acid composition is modified, preferably alabyrinthulid that produces PUFAs only via the elongase-desaturasepathway. the invention provides a method for producing PUFAs using thelabyrinthulid thereof, lipids (microbial oils) containing PUFAs producedusing the method thereof, and uses thereof.

To create a strain containing certain PUFAs other than DHA as the mainfatty acids using labyrinthulids having both the PUFA-PKS pathway andthe elongase-desaturase pathway, first of all, DHA production via thePUFA-PKS pathway needs to be stopped or inhibited by any means.Specifically, methods such as disrupting PUFA-PKS pathway gene withhomologous recombination technique, acquisition of a spontaneous mutantby UV irradiation, drug treatment, or the like, and silencing ofPUFA-PKS genes by RNAi (RNA interference), and the like may beconsidered.

Among these methods, gene-disruption with homologous recombinationtechnique previously described has been technically established.However, the number of drug resistance genes that can be used as markersis generally limited, and there is the problem that when a marker isused in PUFA-PKS pathway gene disruption, the number of markers that canbe used in further gene disruption or gene transfer will decrease. Thismeans a decrease in the number of times that gene-disruption andgene-transfer can be implemented after PUFA-PKS pathway gene isdisrupted, and thus it becomes an obstacle to create higher-performancestrains.

Furthermore, spontaneous mutation and silencing by RNAi are methods witha track record in other organisms but it is unknown whether a desiredstrain in labyrinthulids can be acquired by these methods.

The present invention is directed to provide a labyrinthulid having veryweak or no PUFA-producing activity via the endogenous PUFA-PKS pathwayand having PUFA producing activity via the endogenouselongase-desaturase pathway, a method for producing lipids containingPUFAs produced using the labyrinthulid thereof, and lipids containingPUFAs produced using the labyrinthulid thereof.

Means for Solving the Problems

The present inventors conducted a diligent research, and discovered theexistence of labyrinthulids having very weak or no PUFA producingactivity via the endogenous PUFA-PKS pathway and having PUFA-producingactivity via the endogenous elongase-desaturase pathway. And the presentinvention was accomplished pertaining to microbial oil obtained fromlabyrinthulids thereof, microbial oil-producing labyrinthulids, methodsfor producing microbial oil thereof, and uses of microbial oils.

The gist of the present invention includes the following microbial oils(1) to (28).

(1) Microbial oil satisfying at least one condition selected from thegroup consisting of (a) to (g) below.

(a) ARA is not less than 5% of the total fatty acid composition.

(b) DGLA is not less than 2.5% of the total fatty acid composition.

(c) ETA is not less than 0.35% of the total fatty acid composition.

(d) EPA is not less than 4% of the total fatty acid composition.

(e) n-6 DPA is not greater than 0.20% of the total fatty acidcomposition.

(f) DHA is not greater than 0.50% of the total fatty acid composition.

(g) The total of DHA and n-6 DPA is not greater than 0.7% of the totalfatty acid composition.

(2) The microbial oil according to (1), wherein the microbial oilsatisfies at least one condition selected from the group consisting of(h) to (1) below.

(h) The value of LA/DHA by GC area is not less than 0.6 and not greaterthan 10.

(i) The value of GLA/DHA by GC area is not less than 0.35 and notgreater than 10.

(j) The value of DGLA/DHA by GC area is not less than 0.35 and notgreater than 10.

(k) The value of ARA/DHA by GC area is not less than 0.7 and not greaterthan 50.

(l) The value of EPA/DHA by GC area is not less than 0.35 and notgreater than 50.

(3) The microbial oil according to (1) or (2), wherein the microbial oilsatisfies at least one condition selected from the group consisting of(m) to (o) below.

(m) The value of LA/EPA by GC area is not less than 0.06 and not greaterthan 0.17.

(n) The value of GLA/EPA by GC area is not less than 0.04 and notgreater than 0.12.

(o) The value of DTA/EPA by GC area is not less than 0.01 and notgreater than 0.4.

(4) The microbial oil according to any one of (1) to (3), wherein themicrobial oil satisfies the condition that the value of DTA/ARA by GCarea is not less than 0.01 and not greater than 0.45.

(5) The microbial oil according to any one of (1) to (4), wherein themicrobial oil satisfies the condition that the value of DTADGLA by GCarea is not less than 0.01 and not greater than 1.45.

(6) The microbial oil according to any one of (1) to (5), wherein themicrobial oil satisfies at least one condition selected from the groupconsisting of (p) to (t) below.

(p) The value of LA/n-6 DPA by GC area is not less than 0.4 and notgreater than 20.

(q) The value of GLA/n-6 DPA by GC area is not less than 0.2 and notgreater than 10.

(r) The value of DGLA/n-6 DPA by GC area is not less than 0.35 and notgreater than 30.

(s) The value of ARA/n-6 DPA by GC area is not less than 0.7 and notgreater than 60.

(t) The value of EPA/n-6 DPA by GC area is not less than 1.0 and notgreater than 70.

(7) The microbial oil according to any one of (1) to (6), wherein themicrobial oil satisfies at least one condition selected from the groupconsisting of (u) to (x) below.

(u) The value of DGLA/LA by GC area is not less than 1.4 and not greaterthan 10.

(v) The value of ARA/LA by GC area is not less than 5.1 and not greaterthan 20.

(w) The value of EPA/LA by GC area is not less than 5.5 and not greaterthan 30.

(x) The value of DTA/LA by GC area is not less than 0.01 and not greaterthan 0.4.

(8) The microbial oil according to any one of (1) to (7), wherein themicrobial oil satisfies at least one condition selected from the groupconsisting of (y) and (z) below.

(y) The value of DGLA/GLA by GC area is not less than 4.5 and notgreater than 20.

(z) The value of ARA/GLA by GC area is not less than 9 and not greaterthan 30.

(9) The microbial oil according to any one of (1) to (8), wherein themicrobial oil satisfies the condition that the value of n-6 DPA/DTA byGC area is not greater than 1.5.

(10) The microbial oil according to any one of (1) to (9), wherein themicrobial oil satisfies the condition that the value of DHA/n-3 DPA byGC area is not greater than 4.

(11) The microbial oil according to any one of (1) to (10), wherein themicrobial oil satisfies the condition that the value of C20 PUFA/C22PUFA by GC area is not less than 0.5 and not greater than 50.

(12) The microbial oil according to any one of (1) to (11), wherein themicrobial oil satisfies the condition that the value of n-6 PUFA/n-3PUFA by GC area is not less than 1.8.

(13) A microbial oil obtained from a labyrinthulid genetically modifiedsuch that the fatty acid composition is modified, the labyrinthulidbeing selected from the group consisting of (A) and (B) below.

(A) A labyrinthulid in which the fatty acid composition is modified bydisruption and/or gene silencing.

(B) A labyrinthulid in which the fatty acid composition is modified bytransforming a gene in addition to disruption and/or gene silencing.

(14) The microbial oil according to (13), wherein the disrupted and/orsilenced gene is a PKS gene, a fatty acid elongase gene, and/or a fattyacid desaturase gene.

(15) The microbial oil according to (13) or (14), wherein thetransformed gene is a fatty acid elongase gene and/or a fatty aciddesaturase gene.

(16) The microbial oil according to (14) or (15), wherein the fatty acidelongase gene is the C20 elongase gene.

(17) The microbial oil according to any one of (13) to (16), wherein thefatty acid desaturase gene is the Δ4 desaturase gene and/or the ω3desaturase gene.

(18) The microbial oil according to any one of (13) to (17), wherein themethod for disrupting or transforming a gene of a labyrinthulid iselectroporation, a gene gun method, or gene editing.

(19) The microbial oil according to any one of (13) to (18), wherein themethod for gene silencing of a labyrinthulid is an antisense method orRNA interference.

(20) A microbial oil obtained from a labyrinthulid selected from thegroup consisting of (C) and (D) below.

(C) A labyrinthulid having very weak or no activity of producing PUFAsvia the PUFA-PKS pathway.

(D) A labyrinthulid in which the host PUFA-PKS gene is disrupted orsilenced to a very weak level.

(21) The microbial oil according to (20), wherein the labyrinthulidhaving very weak or no activity of producing PUFAs via the PUFA-PKSpathway is a labyrinthulid belonging to the genus Parietichytrium orgenus Schizochytrium.

(22) The microbial oil according to (21), wherein the labyrinthulidbelonging to the genus Parietichytrium is a labyrinthulid belonging toParietichytrium sarkarianum.

(23) The microbial oil according to (21), wherein the labyrinthulidbelonging to the genus Schizochytrium is a labyrinthulid belonging toSchizochytrium aggregatum.

(24) The microbial oil according to (22), wherein the microorganismbelonging to Parietichytrium sarkarianum is Parietichytrium sp. SEK358(FERM BP-11405), Parietichytrium sarkarianum SEK364 (FERM BP-1298), orParietichytrium sp. SEK517 (FERM BP-11406).

(25) The microbial oil according to (23), wherein the microorganismbelonging to Schizochytrium aggregatum is Schizochytrium aggregatum ATCC28209.

(26) The microbial oil according to (20), wherein the labyrinthulid inwhich the host PUFA-PKS is disrupted or silenced to a very weak levelbelongs to the genus Thraustochytrium.

(27) The microbial oil according to (26), wherein the labyrinthulidbelonging to the genus Thraustochytrium is Thraustochytrium aureum.

(28) A microbial oil that satisfies at least one condition selected fromthe group consisting of (E) to (H) below.

(E) A GC area ratio of ARA after modification is not less than 3 timesgreater than before modification.

(F) A GC area ratio of DGLA after modification is not less than 4 timesgreater than before modification.

(G) A GC area ratio of ETA after modification is not less than 7 timesgreater than before modification.

(H) A GC area ratio of EPA after modification is not less than 7 timesgreater than before modification.

The gist of the present invention includes the following methods forproducing microbial oil (29) to (56).

(29) A method for producing microbial oil satisfying at least onecondition selected from the group consisting of (a) to (g) below.

(a) ARA is not less than 5% of the total fatty acid composition.

(b) DGLA is not less than 2.5% of the total fatty acid composition.

(c) ETA is not less than 0.35% of the total fatty acid composition.

(d) EPA is not less than 4% of the total fatty acid composition.

(e) n-6 DPA is not greater than 0.20% of the total fatty acidcomposition.

(f) DHA is not greater than 0.50% of the total fatty acid composition.

(g) The total of DHA and n-6 DPA is not greater than 0.7% of the totalfatty acid composition.

(30) The method for producing microbial oil according to (29), whereinthe microbial oil satisfies at least one condition selected from thegroup consisting of (h) to (1) below.

(h) The value of LA/DHA by GC area is not less than 0.6 and not greaterthan 10.

(i) The value of GLA/DHA by GC area is not less than 0.35 and notgreater than 10.

(j) The value of DGLA/DHA by GC area is not less than 0.35 and notgreater than 10.

(k) The value of ARA/DHA by GC area is not less than 0.7 and not greaterthan 50.

(l) The value of EPA/DHA by GC area is not less than 0.35 and notgreater than 50.

(31) The method for producing microbial oil according to (29) or (30),wherein the microbial oil satisfies at least one condition selected fromthe group consisting of (m) to (o) below.

(m) The value of LA/EPA by GC area is not less than 0.06 and not greaterthan 0.17.

(n) The value of GLA/EPA by GC area is not less than 0.04 and notgreater than 0.12.

(o) The value of DTA/EPA by GC area is not less than 0.01 and notgreater than 0.4.

(32) The method for producing microbial oil according to any one of (29)to (31), wherein the microbial oil satisfies the condition that thevalue of DTA/ARA by GC area is not less than 0.01 and not greater than0.45.

(33) The method for producing microbial oil according to any one of (29)to (32), wherein the microbial oil satisfies the condition that thevalue of DTA/DGLA by GC area is not less than 0.01 and not greater than1.8.

(34) The method for producing microbial oil according to any one of (29)to (33), wherein the microbial oil satisfies at least one conditionselected from the group consisting of (p) to (t) below.

(p) The value of LA/n-6 DPA by GC area is not less than 0.4 and notgreater than 20.

(q) The value of GLA/n-6 DPA by GC area is not less than 0.2 and notgreater than 10.

(r) The value of DGLA/n-6 DPA by GC area is not less than 0.35 and notgreater than 30.

(s) The value of ARA/n-6 DPA by GC area is not less than 0.7 and notgreater than 60.

(t) The value of EPA/n-6 DPA by GC area is not less than 0.4 and notgreater than 70.

(35) The method for producing microbial oil according to any one of (29)to (34), wherein the microbial oil satisfies at least one conditionselected from the group consisting of (u) to (x) below.

(u) The value of DGLALA by GC area is not less than 1.4 and not greaterthan 10.

(v) The value of ARA/LA by GC area is not less than 5.1 and not greaterthan 20.

(w) The value of EPA/LA by GC area is not less than 5.5 and not greaterthan 30.

(x) The value of DTA/LA by GC area is not less than 0.01 and not greaterthan 0.4.

(36) The method for producing microbial oil according to any one of (29)to (35), wherein the microbial oil satisfies at least one conditionselected from the group consisting of (y) and (z) below.

(y) The value of DGLA/GLA by GC area is not less than 4.5 and notgreater than 20.

(z) The value of ARA/GLA by GC area is not less than 9 and not greaterthan 30.

(37) The method for producing microbial oil according to any one of (29)to (36), wherein the microbial oil has a value of n-6 DPA/DTA by GC areaof not greater than 1.5.

(38) The method for producing microbial oil according to any one of (29)to (37), wherein the microbial oil has a value of DHA/n-3 DPA by GC areaof not greater than 4.

(39) The method for producing microbial oil according to any one of (29)to (38), wherein the microbial oil satisfies the condition that thevalue of C20 PUFA/C22 PUFA by GC area is not less than 0.5 and notgreater than 50.

(40) The method for producing microbial oil according to any one of (29)to (39), wherein the microbial oil satisfies the condition that thevalue of n-6 PUFA/n-3 PUFA by GC area is not less than 1.8.

(41) A method for producing microbial oil whereby microbial oil iscaused to be produced in a labyrinthulid genetically modified such thatthe fatty acid composition is modified, the labyrinthulid being selectedfrom the group consisting of (A) and (B) below.

(A) A labyrinthulid in which the fatty acid composition is modified bygene-disruption and/or gene silencing.

(B) A labyrinthulid in which the fatty acid composition is modified bytransforming a gene in addition to disruption and/or gene silencing.

(42) The method for producing microbial oil according to (41), whereinthe disrupted and/or silenced gene is a PKS gene, a fatty acid elongasegene, and/or a fatty acid desaturase gene.

(43) The method for producing microbial oil according to (41) or (42),wherein the transformed gene is a fatty acid elongase gene and/or afatty acid desaturase gene.

(44) The method for producing microbial oil according to (42) or (43),wherein the fatty acid elongase gene is the C20 elongase gene.

(45) The method for producing microbial oil according to any one ofclaims 41 to 44, wherein the fatty acid desaturase gene is the Δ4desaturase gene and/or the ω3 desaturase gene.

(46) The method for producing microbial oil according to any one of (41)to (45), wherein the method for disrupting or transforming a gene of alabyrinthulid is electroporation, a gene gun method, or gene editing.

(47) The method for producing microbial oil according to any one of (41)to (46), wherein the method for gene silencing of a labyrinthulid is anantisense method or RNA interference.

(48) A method for producing microbial oil, wherein microbial oil iscaused to be produced in a labyrinthulid selected from the groupconsisting of (C) and (D) below.

(C) A labyrinthulid having very weak or no activity of producing PUFAsvia the PUFA-PKS pathway.

(D) A labyrinthulid in which the host PUFA-PKS gene is disrupted orsilenced to a very weak level.

(49) The method for producing microbial oil according to (48), whereinthe labyrinthulid having very weak or no activity of producing PUFAs viathe PUFA-PKS pathway is a labyrinthulid belonging to the genusParietichytrium or genus Schizochytrium.

(50) The method for producing microbial oil according to (49), whereinthe labyrinthulid belonging to the genus Parietichytrium is alabyrinthulid belonging to Parietichytrium sarkarianum.

(51) The method for producing microbial oil according to (50), whereinthe labyrinthulid belonging to the genus Schizochytrium is alabyrinthulid belonging to Schizochytrium aggregatum.

(52) The method for producing microbial oil according to (50), whereinthe microorganism belonging to Parietichytrium sarkarianum isParietichytrium sp. SEK358 (FERM BP-11405), Parietichytrium sarkarianumSEK364 (FERM BP-11298), or Parietichytrium sp. SEK517 (FERM BP-11406).

(53) The method for producing microbial oil according to (51), whereinthe microorganism belonging to Schizochytrium aggregatum isSchizochytrium aggregatum ATCC 28209.

(54) The method for producing microbial oil according to (48), whereinthe labyrinthulid in which the host PUFA-PKS gene is disrupted orsilenced to a very weak level belongs to the genus Thraustochytrium.

(55) The method for producing microbial oil according to (54), whereinthe labyrinthulid belonging to the genus Thraustochytrium isThraustochytrium aureum.

(56) A method for producing microbial oil satisfying at least onecondition selected from the group consisting of (E) to (H) below.

(E) The GC area ratio of ARA after modification is not less than 3 timesgreater than before modification.

(F) The GC area ratio of DGLA after modification is not less than 4times greater than before modification.

(G) The GC area ratio of ETA after modification is not less than 7 timesgreater than before modification.

(H) The GC area ratio of EPA after modification is not less than 7 timesgreater than before modification.

Furthermore, the gist of the present invention includes the followingfood, animal feed, medication, or industrial product (57), the followinggenetically modified labyrinthulid (58), and the following method forcreating the genetically modified labyrinthulid (59).

(57) A food, animal feed, medication, or industrial product includingthe microbial oil described in any one of (1) to (28) as a lipidcomposition.

(58) A labyrinthulid genetically modified such that a produced fattyacid composition is modified, the labyrinthulid producing the microbialoil described in any one of (1) to (28).

(59) A method for creating the labyrinthulid genetically modified suchthat the produced fatty acid composition described in (58) is modified.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the elongase-desaturase pathway and enzymes relatedto this biosynthesis pathway. (Brief description of symbols) C16:0:Palmitic acid; C18:0: Stearic acid; C18:n-9: Oleic acid; C18:2n-6:Linoleic acid (LA); C18:3n-3: α-Linolenic acid (ALA); C18:3n-6:γ-Linolenic acid (GLA); C18:4n-3: Stearidonic acid (STA); C20:2n-6:Eicosadienoic acid (EDA); C20:3n-3: Eicosatrienoic acid (ETrA);C20:3n-6: Dihomo-γ-linolenic acid (DGLA); C20:4n-3: Eicosatetraenoicacid (ETA); C20:4n-6: Arachidonic acid (ARA); 20:5n-3: Eicosapentaenoicacid (EPA): C22:4n-6: Docosatetraenoic acid (DTA); C22:5n-3: n-3Docosapentaenoic acid (n-3 DPA); C22:5n-6: n-6 Docosapentaenoic acid(n-6 DPA); C22:6n-3: Docosahexaenoic acid (DHA); C16e: C16 elongase;Δ9d: Δ9 desaturase; Δ12d: Δ12 desaturase; Δ15d: Δ15 desaturase; Δ9e: Δ9elongase; Δ6d: Δ6 desaturase; Δ8d: Δ8 desaturase; C18e: C18 elongase:Δ5d: Δ5 desaturase; C20e: C20 elongase; Δ17d: Δ17 desaturase; Δ4d: Δ4desaturase. Δ15 desaturase and Δ17 desaturase are each sometimes calledω3 desaturase.

FIG. 2 illustrates a plasmid containing an SV40 terminator sequencederived from a subcloned pcDNA 3.1 Myc-His vector.

FIG. 3 is a schematic diagram of the primers used in fusion PCR and theproducts. The final product is a fused sequence of an ubiquitin promoterderived from Thraustochytrium aureum ATCC 34304 and an artificiallysynthesized neomycin resistance gene.

FIG. 4 illustrates the BgII cassette of the produced artificiallysynthesized neomycin resistance gene.

FIG. 5 is a schematic diagram of the primers used in fusion PCR and theproducts. The final product is a fused sequence of an ubiquitin promoterderived from Thraustochytrium aureum ATCC 34304 and a hygromycinresistance gene derived from pcDNA 3.1/Hygro.

FIG. 6 illustrates the BglII cassette of the produced hygromycinresistance gene derived from pcDNA 3.1/Hygro.

FIG. 7 illustrates a plasmid containing a C20 elongase sequence ofcloned genus Parietichytrium.

FIG. 8 illustrates a plasmid in which a BglII site has been inserted ina C20 elongase sequence of genus Parietichytrium of the plasmidillustrated in FIG. 7.

FIG. 9 illustrates the produced genus Parietichytrium C20 elongase genetargeting vectors (two types). As a drug resistance marker, the vectorshave a neomycin resistance gene (pRH85) or a hygromycin resistance gene(pRH86).

FIG. 10 is a schematic diagram illustrating the positions of the PCRprimers used in identification of a C20 elongase gene disruption strainof Parietichytrium sarkarianum SEK364, and the expected product.

FIG. 11 illustrates an evaluation of C20 elongase gene disruption by PCRusing Parietichytrium sarkarianum SEK364 genome DNA as a template.

Description of Symbols

+/+: Parietichytrium sarkarianum SEK364 wild-type strain;

+/−: C20 elongase gene first allele homologous recombinant derived fromParietichytrium sarkarianum SEK364:

−/−: C20 elongase disruption strain derived from Parietichytriumsarkarianum SEK364

FIG. 12 illustrates a comparison of fatty acid compositions of theParietichytrium sarkarianum SEK364 wild-type strain and the C20 elongasegene disruption strain. The black bars and white bars represent thefatty acid composition of the wild-type strain and the gene disruptionstrain, respectively. The values are mean±standard deviation.

FIG. 13 lists the fatty acid proportions of the C20 elongase genedisruption strain when the Parietichytrium sarkarianum SEK364 wild-typestrain is taken as 100%.

FIG. 14 illustrates a plasmid containing a cloned Parietichytrium sp.SEK571 Δ4 desaturase gene sequence and the peripheral sequence.

FIG. 15 illustrates a plasmid in which the Parietichytrium sp. SEK571 Δ4desaturase gene sequence and a 600 bp downstream of the Δ4 desaturasegene sequence have been deleted from the plasmid illustrated in FIG. 14and a BglII site has been inserted.

FIG. 16 illustrates a plasmid in which a DNA fragment containing anartificially synthesized neomycin resistance gene cassette has beenbound at the BglII site of the plasmid illustrated in FIG. 15.

FIG. 17 illustrates the produced genus Parietichytrium Δ4 desaturasegene targeting vector. As a drug resistance marker, the vector has aneomycin resistance gene.

FIG. 18 is a schematic diagram illustrating the positions of the PCRprimers used in identification of a Δ4 desaturase gene disruption strainof a genus Parietichytrium labyrinthulid, and the expected product(primers are set within the homologous recombination region).

FIG. 19 illustrates evaluation results of Δ4 desaturase gene disruptionby PCR using Parietichytrium sarkarianum SEK364 genome DNA as atemplate.

FIG. 20 is a gas chromatograph analysis chart of the fatty acidcompositions of the Parietichytrium sarkarianum SEK364 wild-type strainand the Δ4 desaturase gene disruption strain thereof.

FIG. 21 is a partial enlarged diagram of FIG. 20.

FIG. 22 shows a comparison of the fatty acid compositions of theParietichytrium sarkarianum SEK364 wild-type strain and the Δ4desaturase gene disruption strain thereof. This table is aquantification of the chart of FIG. 20. In the table, the less-than sign(<) indicates less than or equal to the number following it.

FIG. 23 illustrates an evaluation of C20 elongase gene disruption by PCRusing Parietichytrium sp. SEK358 strain genome DNA as a template.

Description of Symbols

+/+: Parietichytrium sp. SEK358 wild-type strain;

−/−: C20 elongase gene disruption strain derived from Parietichytriumsp. SEK358 strain

FIG. 24 illustrates a comparison of fatty acid compositions of theParietichytrium sp. SEK358 wild-type strain and the C20 elongase genedisruption strain derived from Parietichytrium sp. SEK358 strain. Thewhite bars and black bars represent the fatty acid composition of thewild-type strain and the gene disruption strain, respectively.

FIG. 25 lists the fatty acid proportions of the C20 elongase genedisruption strain derived from the Parietichytrium sp. SEK358 strainwhen the Parietichytrium sp. SEK358 wild-type strain is taken as 100%.In the Parietichytrium sp. SEK358 wild-type strain, cases where therelevant fatty acid is below the detection limit are indicated by adiagonal line.

FIG. 26 is a schematic diagram illustrating the positions of the PCRprimers used in identification of a Δ4 desaturase gene disruption strainof a genus Parietichytrium labyrinthulid, and the expected product(primers are set outside the homologous recombination region).

FIG. 27 illustrates evaluation results of Δ4 desaturase gene disruptionby PCR using Parietichytrium sp. SEK358 genome DNA as a template in thecase where the primers are set within the homologous recombinationregion.

FIG. 28 illustrates evaluation results of Δ4 desaturase gene disruptionby PCR using Parietichytrium sp. SEK358 genome DNA as a template in thecase where the primers are set outside the homologous recombinationregion.

FIG. 29 is a gas chromatograph analysis chart of the fatty acidcompositions of the Parietichytrium sp. SEK358 wild-type strain and theΔ4 desaturase gene disruption strain thereof.

FIG. 30 is a partial enlarged diagram of FIG. 29.

FIG. 31 shows a comparison of the fatty acid compositions of theParietichytrium sp. SEK358 wild-type strain and the Δ4 desaturase genedisruption strain thereof. This table is a quantification of the chartof FIG. 29. In the table, the less-than sign (<) indicates less than orequal to the number following it.

FIG. 32 illustrates an evaluation of C20 elongase gene disruption by PCRusing Parietichytrium sp. SEK571 strain genome DNA as a template.

Description of Symbols

+/+: Parietichytrium sp. SEK571 wild-type strain;

−/−: C20 elongase gene disruption strain derived from Parietichytriumsp. SEK571 strain

FIG. 33 illustrates a comparison of fatty acid compositions of theParietichytrium sp. SEK571 wild-type strain and the C20 elongase genedisruption strain derived from Parietichytrium sp. SEK571 strain. Thewhite bars and black bars represent the fatty acid composition of thewild-type strain and the gene disruption strain, respectively.

FIG. 34 lists the fatty acid proportions of the C20 elongase genedisruption strain derived from the Parietichytrium sp. SEK571 strainwhen the Parietichytrium sp. SEK571 wild-type strain is taken as 100%.

FIG. 35 illustrates results of RACE in which an elongase gene derivedfrom T. aureum ATCC 34304 is amplified in Comparative Example 1-2.(Brief description of symbols)

1: 5′-RACE using synthetic adapter-specific oligonucleotide anddegenerate oligonucleotide elo-R;

2: 3′-RACE using synthetic adapter-specific oligonucleotide anddegenerate oligonucleotide elo-F;

3: 5′-RACE using only elo-R (negative control);

4: 3′-RACE using only elo-F (negative control);

5: 5′-RACE using only synthetic adapter-specific oligonucleotide(negative control); 6: 3′-RACE using only synthetic adapter-specificoligonucleotide (negative control)

FIG. 36 illustrates an evaluation of a transformant into which KONeorwas transformed in Comparative Example 1-7.

(A) illustrates the oligonucleotide primer pair used in evaluation ofthe transformant by PCR using genome DNA as a template. (Briefdescription of symbols)

(1) Neor detection primers (SNeoF and SNeoR);

(2) KO confirmation 1 (KO Pro F SmaI and KO Term R SmaI);

(3) KO confirmation 2 (E2 KO ProF EcoRV and SNeoR):

(4) KO confirmation 3 (SNeoF and E2 KO Term R EcoRV);

(5) TaELO2 detection (E2 HindIII and E2 XbaI).

(B) illustrates the agarose electrophoresis diagram in evaluation of thetransformant by PCR using genome DNA as a template. (Brief descriptionof symbols)

1, 5, 9, 13, 17: Transformant;

2, 6, 10, 14, 18: Wild-type strain;

3, 7, 11, 15, 19: Using KONeor as a template:

4, 8, 12, 16: No template.

Furthermore, the used oligonucleotide primer pairs (1) to (5) are thelane numbers.

FIG. 37 illustrates the results of confirmation of TaELO2 copy number bysouthern blotting in Comparative Example 1-8. (Brief description ofsymbols)

1: Genome DNA (2.5 μg), BamHI treatment;

2: BglII treatment;

3: EcoRI treatment;

4: EcoRV treatment;

5: HindIII treatment;

6: KpnI treatment;

7: SmaI treatment;

8: XbaI treatment;

9: Positive control (PCR product amplified with 1 ng of E2 KO Pro FEcoRV and E2 KO Term R EcoRV. Including TaELO2.)

FIG. 38 illustrates an evaluation by southern blotting of a transformantinto which TKONeor was transformed in Comparative Example 1-9.

(A) is a schematic diagram of southern blotting to detect a wild-typeallele or mutant allele by TKONeor transfer.

(B) illustrates the results of southern blotting. (Brief description ofsymbols)

1: T. aureum wild-type strain (2.5 μg of genome DNA);

2, 3: TKONeor transfer transformant (2.5 μg of genome DNA);

4: Positive control (PCR product amplified with 50 ng of E2 KO ProFEcoRV and E2 KO Term R EcoRV. Includes TaELO2.)

FIG. 39 illustrates an evaluation by PCR using as a template genome DNAof a transformant obtained by retransfer of KOub600Hygr in ComparativeExample 1-11.

(A) illustrates the oligonucleotide primer pair used. (Brief descriptionof symbols)

(1) TaELO2 ORF detection (SNeoF and SNeoR):

(2) KO confirmation (E2 KO Pro F EcoRV and ubi-hygro R)

(B) illustrates the agarose electrophoresis diagram of PCR usingoligonucleotide primer pair (1) of KO confirmation. The arrows indicatethe transformant of which specific product amplification was confirmedand which was estimated to be TaELO2 deletion homozygote.

(C) illustrates the agarose electrophoresis diagram of PCR usingoligonucleotide primer pair (2) of TaELO2 ORF detection in atransformant identified as a TaELO2 deletion homozygote. (Briefdescription of symbols)

1: Using KOub600Hygr as a template;

2: Wild-type strain

FIG. 40 illustrate an evaluation by southern blotting of a transformantobtained by retransfer of KOub600Hygr in Comparative Example 1-11.

(A) is a schematic diagram of southern blotting to detect a wild-typeallele, a mutant allele by KONeor transfer, and a mutant allele byKOub600Hygr transfer.

(B) illustrates the results of southern blotting. (Brief description ofsymbols)

1, 9: Wild-type strain;

2 to 8 and 10 to 16: TaELO2 deletion homozygote

FIG. 41 illustrates the results of southern blotting to detect TaELO2 inComparative Example 1-11. (Brief description of symbols)

1: Wild-type strain;

2 to 5: T TaELO2 deletion homozygote

FIG. 42 illustrates the results of agarose electrophoresis of RT-PCR todetect TaELO2 mRNA in Comparative Example 1-11. (Brief description ofsymbols)

1 to 4: TaELO2 deletion homozygote;

5: Wild-type strain; 6 to 9: TaELO2 deletion homozygote, using total RNAas a template (negative control);

10: Wild-type strain, using total RNA as a template (negative control);

11: Using wild-type genome DNA as a template (positive control)

FIG. 43 illustrates the results of a fatty acid composition comparisonof the wild-type strain and TaELO2 deletion homozygote in ComparativeExample 1-12.

FIG. 44 is a schematic diagram of the primers used in fusion PCR and theproducts. The final product is a fused sequence of 18S rDNA derived fromThraustochytrium aureum ATCC 34304, an EF1α promoter derived fromThraustochytrium aureum ATCC 34304, an artificially synthesized neomycinresistance gene, and an EF1α terminator derived from Thraustochytriumaureum ATCC 34304.

FIG. 45 illustrates a plasmid in which a portion of the ligated DNAfragment in FIG. 42 was cloned. The plasmid contains a partial sequenceof the 3′ side from the EcoRI site of 18S rDNA derived fromThraustochytrium aureum ATCC 34304, an EF1α promoter derived fromThraustochytrium aureum ATCC 34304, an artificially synthesized neomycinresistance gene, and a partial sequence of the 5′ side from the NcoIsite of the EF1α terminator derived from Thraustochytrium aureum ATCC34304.

FIG. 46 illustrates the produced targeting vector of Thraustochytriumaureum ATCC 34304 PKS pathway associated gene orfA. As a drug resistancemarker, the vector has a neomycin resistance gene.

FIG. 47 illustrates a plasmid containing an upstream sequence of theThraustochytrium aureum ATCC 34304 PKS pathway associated gene orfA, aubiquitin promoter derived from Thraustochytrium aureum ATCC 34304, anda hygromycin resistance gene.

FIG. 48 illustrates the produced targeting vector of Thraustochytriumaureum ATCC 34304 PKS pathway associated gene orfA. As a drug resistancemarker, the vector has a hygromycin resistance gene.

FIG. 49 is a schematic diagram illustrating the positions of thesouthern hybridization analysis probes used in identification of the PKSpathway associated gene orfA disruption strain of Thraustochytriumaureum ATCC 34304, and the expected size of the gene fragment.

FIG. 50 illustrates an evaluation of PKS pathway associated gene orfAdisruption by southern hybridization using Thraustochytrium aureum ATCC34304 genome DNA. (Brief description of symbols)

T. au: Thraustochytrium aureum ATCC 34304 wild-type strain;

+/−: PKS pathway associated gene orfA first allele homologousrecombinant derived from Thraustochytrium aureum ATCC 34304;

−/−: PKS pathway associated gene orfA disruption strain derived fromThraustochytrium aureum ATCC 34304

FIG. 51 illustrates a comparison of fatty acid compositions of theThraustochytrium aureum ATCC 34304 wild-type strain and the PKS pathwayassociated gene orfA disruption strain. The white bars and black barsrepresent the fatty acid composition of the wild-type strain and thegene disruption strain, respectively. The values are mean±standarddeviation.

FIG. 52 lists the fatty acid proportions of the PKS pathway associatedgene orfA disruption strain when the Thraustochytrium aureum ATCC 34304wild-type strain is taken as 100%.

FIG. 53 is a schematic diagram of the primers used in fusion PCR and theproducts. The final product is a fused sequence of an ubiquitin promoterderived from Thraustochytrium aureum ATCC 34304 and a blasticidinresistance gene derived from pTracer-CMV/Bsd/lacZ.

FIG. 54 illustrates the produced BglII cassette of the blasticidinresistance gene derived from pTracer-CMV/Bsd/lac.

FIG. 55 is a schematic diagram of the primers used in fusion PCR and theproducts. The final product is a fused sequence of an ubiquitin promoterderived from Thraustochytrium aureum ATCC 34304 and an enhanced GFP gene(available from Clontech Laboratories, Inc.).

FIG. 56 is a schematic diagram of the primers used in fusion PCR and theproducts. The final product is a fused sequence of an ubiquitin promoterderived from Thraustochytrium aureum ATCC 34304, an enhanced GFP gene(available from Clontech Laboratories, Inc.), and a zeocin resistancegene derived from pcDNA 3.1 Zeo(+).

FIG. 57 illustrates the produced BglII cassette of the enhancedGFP-zeocin resistance fusion gene.

FIG. 58 illustrates a plasmid containing a cloned Thraustochytriumaureum ATCC 34304 C20 elongase sequence and the peripheral sequence.

FIG. 59 illustrates a plasmid in which the Thraustochytrium aureum ATCC34304 C20 elongase sequence has been completely deleted from the plasmidillustrated in FIG. 56 and a BglII site has been inserted.

FIG. 60 illustrates the produced Thraustochytrium aureum ATCC 34304 C20elongase gene targeting vectors (two types). As a drug resistancemarker, the vectors have a blasticidin resistance gene (pRH43) or anenhanced GFP-zeocin resistance fusion gene (pRH54).

FIG. 61 is a schematic diagram illustrating the position of the southernhybridization analysis probe used in identification of the C20 elongasegene disruption strain of the Thraustochytrium aureum ATCC 34304 PKSpathway (orfA gene) disruption strain, and the expected size of the genefragment.

FIG. 62 illustrates an evaluation of C20 elongase gene disruption bysouthern hybridization using Thraustochytrium aureum ATCC 34304 genomeDNA. (Brief description of symbols)

T. au: Thraustochytrium aureum ATCC 34304 wild-type strain:

−/−: PKS pathway (orfA gene) and C20 elongase gene double disruptionstrain derived from Thraustochytrium aureum ATCC 34304

FIG. 63 illustrates a comparison of fatty acid compositions of theThraustochytrium aureum ATCC 34304 wild-type strain and the PKS pathway(orfA gene) and C20 elongase gene double disruption strain. The whitebars and black bars represent the fatty acid composition of thewild-type strain and the gene disruption strain, respectively. Thevalues are mean±standard deviation.

FIG. 64 lists the fatty acid proportions of the PKS pathway (orfA gene)and C20 elongase gene double disruption strain when Thraustochytriumaureum ATCC 34304 wild-type strain is taken as 100%.

FIG. 65 illustrates a plasmid containing from 1071 bp upstream of the Δ4desaturase gene to 1500 bp within the Δ4 desaturase gene of a clonedThraustochytrium aureum ATCC 34304 strain.

FIG. 66 illustrates a plasmid in which a sequence of 60 bp upstream ofthe Δ4 desaturase gene of the plasmid illustrated in FIG. 63 and asequence of 556 bp containing the start codon within the Δ4 desaturasegene (616 bp, SEQ ID NO: 205) have been deleted and a BglII site hasbeen inserted in the deleted portion.

FIG. 67 illustrates the produced Thraustochytrium aureum ATCC 34304strain Δ4 desaturase gene targeting vectors (two types). As a drugresistance marker, the vectors have a blasticidin resistance gene (pTM6)or an enhanced GFP-zeocin resistance fusion gene (pTM8).

FIG. 68 is a schematic diagram illustrating the positions of the PCRprimers used in identification of the Δ4 desaturase gene disruptionstrain of the Thraustochytrium aureum ATCC 34304 PKS pathway (orfA gene)disruption strain, and the expected products.

FIG. 69 illustrates an evaluation of Δ4 desaturase gene disruption byPCR using Thraustochytrium aureum ATCC 34304 strain genome DNA as atemplate. (Brief description of symbols)+/+: PKS pathway (orfA gene)disruption strain derived from Thraustochytrium aureum ATCC 34304; +/−:Δ4 desaturase first allele homologous recombinant derived from PKSpathway (orfA gene) disruption strain derived from Thraustochytriumaureum ATCC 34304: −/−: PKS pathway (orfA gene) and Δ4 desaturase genedouble disruption strain derived from Thraustochytrium aureum ATCC 34304

FIG. 70 illustrates a comparison of fatty acid compositions of theThraustochytrium aureum ATCC 34304 wild-type strain and the PKS pathway(orfA gene) and Δ4 desaturase gene double disruption strain. The whitebars and black bars represent the fatty acid composition of thewild-type strain and the gene disruption strain, respectively.

FIG. 71 lists the fatty acid proportions of the PKS pathway (orfA gene)and Δ4 desaturase gene double disruption strain when theThraustochytrium aureum ATCC 34304 wild-type strain is taken as 100%.

MODE FOR CARRYING OUT THE INVENTION

The opportunity for the present invention came about due to thediscovery of a new pattern of biosynthesis pathway of polyunsaturatedfatty acids (PUFAs) in microorganisms called labyrinthulids.Specifically, it is known that PUFAs are generally biosynthesized viathe elongase-desaturase pathway, but in some organisms. PUFAs are alsobiosynthesized via another pathway called the PUFA-PKS pathway. In thepast, two types of labyrinthulids were found to exist, namely (I) thetype that produces only via the PUFA-PKS pathway and (II) the type thatproduces via the elongase-desaturase pathway and PUFA-PKS pathway. Thistime, a third type, (II) a type that produces only via theelongase-desaturase pathway, was newly discovered. In short, a new“pattern” of PUFA biosynthesis pathway was discovered.

Examples are as follows. (1) Labyrinthulids having very weak or noPUFA-producing activity via the endogenous PUFA-PKS pathway and havingPUFA-producing activity via the endogenous elongase-desaturase pathway.The present invention relates to labyrinthulids of the above (III) typethat produces only via the elongase-desaturase pathway. Theselabyrinthulids encompass labyrinthulids of type (III) produced byisolating, culturing, and amplifying a wild-type strain having a PUFAbiosynthesis pathway, and labyrinthulids having “very weak or no”PUFA-producing activity via the PUFA-PKS pathway.

(2) The labyrinthulids according to (1) above that do not have theendogenous PUFA-PKS pathway. The present invention is limited to (1)above, which are labyrinthulids having no genes or enzymes themselvesthat constitute the PUFA-PKS pathway.

(3) The labyrinthulids according to (1) above that have very weak or noendogenous PUFA-PKS pathway activity. The present invention is limitedto (1) above that have information related to genes of enzymesconstituting the PUFA-PKS pathway on the genome but do not express them(and therefore have no activity), or that express them only very weaklyor not at all.

(4) The labyrinthulids according to any of (1) to (3) above, wherein DHAand/or n-6 DPA production activity has been lost or the producedquantity of DHA and/or n-6 DPA has been markedly decreased by endogenousΔ4 desaturase gene disruption.

Labyrinthulids of type (III) which produce PUFAs only via theelongase-desaturase pathway differ from those of type (I) which produceonly via the PUFA-PKS pathway and those of type (IT) which produce viathe elongase-desaturase pathway and PUFA-PKS pathway in that they haveno PUFA-PKS pathway. Based on this viewpoint, the present inventiondefines labyrinthulids of the type that produces only via theelongase-desaturase pathway. In other words, the above endogenous Δ4desaturase gene disruption can be considered a method for determiningwhether a labyrinthulid is of the type that produces only via theelongase-desaturase pathway.

(5) The labyrinthulids according to (1) to (3) above, wherein DHA and/orn-6 DPA production activity has been lost or the produced quantity ofDHA and/or n-6 DPA has been markedly decreased through endogenous C20elongase gene disruption.

Labyrinthulids of type (III) which produce only via theelongase-desaturase pathway differ from those of type (I) which produceonly via the PUFA-PKS pathway and those of type (II) which produce viathe elongase-desaturase pathway and PUFA-PKS pathway in that they haveno PUFA-PKS pathway. Based on this viewpoint, the present inventiondefines labyrinthulids of the type that produces only via theelongase-desaturase pathway. In other words, the above endogenous C20elongase gene disruption can be considered a method for determiningwhether a labyrinthulid is of the type that produces only via theelongase-desaturase pathway.

(6) The labyrinthulids according to (1) to (5) above that is amicroorganism belonging to either the genus Parietichytrium or the genusSchizochytrium.

Labyrinthulids belonging to the genus Parietichytrium or the genusSchizochytrium were known before the filing date of the presentapplication, but it was not known that they are labyrinthulids of type(III) which produce PUFAs only via the elongase-desaturase pathway.

(7) The labyrinthulids according to (6) above, wherein the microorganismis Parietichytrium sp. SEK358 (FERM BP-11405), Parietichytriumsarkarianum SEK364 (FERM BP-11298), Parietichytrium sp. SEK517 (FERMBP-1406), or Schizochytrium aggregatum ATCC 28209.

(8) A method for producing lipids containing PUFAs, the method includingculturing the labyrinthulids described in any of (1) to (7) above in aculture medium and collecting the lipids from the culture.

(9) A method for producing lipids containing PUFAs, the method includingculturing labyrinthulids, that were transformed using the labyrinthulidsdescribed in any of (1) to (7) above as hosts with the objective ofmodifying the fatty acid composition and/or highly accumulating fattyacids, in a culture medium and collecting the lipids from the culture.

(10) Lipids containing PUFAs, the lipids being produced by the method of(8) or (9) above.

The present invention can provide labyrinthulea that produce PUFAs viaonly the elongase-desaturase pathway.

The creation of labyrinthulids equivalent to type (III) which produceonly via the elongase-desaturase pathway is also possible, byspontaneous mutation or genetic recombination from type (II) whichproduce via the elongase-desaturase pathway and PUFA-PKS pathway.Examples of these include microorganisms belonging to the genusThraustochytrium. By using these microorganisms, it is possible toobtain the same polyunsaturated fatty acids as by labyrinthulids of type(III).

[Microorganisms]

Labyrinthulids having very weak or no activity of producing PUFAs viathe PUFA-PKS pathway includes labyrinthulids that produce PUFAs only viathe elongase-desaturase pathway. A labyrinthulid having very weakPUFA-producing activity means a labyrinthulid in which theelongase-desaturase pathway gene has been disrupted, and which cannotproduce PUFAs, and cannot be cultured without supplementing the culturemedium with PUFAs. It means that not greater than 1/100 of the DHAsynthesized in the organism is DHA synthesized via the PUFA-PKS pathway.The labyrinthulid that has very weak or no activity of producing PUFAsvia the endogenous PUFA-PKS pathway and is capable of producing PUFAsvia the endogenous elongase-desaturase pathway is not particularlylimited, but particularly preferred examples are labyrinthulidsbelonging to the genus Parietichytrium or the genus Schizochytrium.Particularly preferred among these are Parietichytrium sp. SEK358 (FERMBP-1405). Parietichytrium sarkarianum SEK364 (FERM BP-11298),Parietichytrium sp. SEK517 (FERM BP-11406), or Schizochytrium aggregatumATCC 28209.

Parietichytrium sp. SEK358 was obtained by the method described below.First, 10 mL of surface water collected in the Ishigakijima-Miyaragawaestuary region was placed in a test tube. Pine pollen was added, and thetest tube was left to stand at room temperature. After 7 days, asterilized agar culture medium (2 g of glucose, 1 g of peptone, 0.5 g ofyeast extract, 0.2 g of chloramphenicol, 15 g of agar, 100 mL ofdistilled water, 900 mL of sea water) was swabbed with this pine pollen,and colonies that emerged after 5 days were separated and cultured. Thiswas repeated several times, and cells were separated. This strain wasinternationally deposited on Aug. 11, 2011 as accession number FERMBP-11405 at the International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology (Tsukuba Central6, 1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture), and can be procuredtherefrom.

Parietichytrium sarkarianum SEK364 was obtained by culturing a sea watersample collected in the Ishigakijima-Fukidogawa estuary region andseparating cells in the same manner as above. This strain wasinternationally deposited on Sep. 24, 2010 as accession number FERMBP-11298 at the International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology (Tsukuba Central6, 1-1-1 Higashi, Tsukuba City. Ibaraki Prefecture), and can be procuredtherefrom.

Parietichytrium sp. SEK571 was obtained by culturing a sea water samplecollected in the Iriomotejima-Shiiragawa estuary region and separatingcells in the same manner as above. This strain was internationallydeposited on Aug. 11, 2011 as accession number FERM BP-11406 at theInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology (Tsukuba Central 6, 1-1-1 Higashi,Tsukuba City, Ibaraki Prefecture), and can be procured therefrom.

Schizochytrium aggregatum ATCC 28209 has been deposited at ATCC, and canbe procured therefrom. Labyrinthulids in which PUFA-PKS has beendisrupted or expression has been inhibited to a very weak level includelabyrinthulids that produce PUFAs via the elongase-desaturase pathwayand PUFA-PKS pathway. The PUFA-PKS pathway is required to exist inlabyrinthulids that produce PUFAs only via the PUFA-PKS pathway, andwhen disrupted, there is a requirement for PUFAs. Labyrinthulids thatproduce PUFAs via the elongase-desaturase pathway and PUFA-PKS pathway,even in a case where the PUFA-PKS pathway has been disrupted, differ inthat there is no requirement for PUFAs.

Disruption of the Δ4-desaturase gene using the present invention candramatically reduce the produced quantities of DHA and n-6 DPA whilemaintaining the produced quantity of PUFAs overall.

The properties of the microorganisms obtained by the present inventionmay be combined as desired.

[Microbial Oil]

The present invention relates to microbial oil containing the fatty acidprofile of the present invention. The microorganism of the presentinvention contains not less than 15 wt. %, preferably not less than 30wt. %, more preferably not less than 50 wt. %, and even more preferablynot less than 70 wt. % of lipid components per gram of cells. The lipidcomponents contain not less than 30 wt. %, preferably not less than 50wt. %, and more preferably not less than 70 wt. % of fatty acidcomponents. The fatty acid components accumulate as not less than 70 wt.%, preferably not less than 80 wt. %, and more preferably not less than90 wt. % of triglycerides. The microbial oil of the present invention is“crude oil” or “refined oil” containing at least approximately 35 wt. %of triacylglycerol fraction. “Crude oil” is oil extracted from microbialbiomass which has not undergone further treatment. “Refined oil” is oilobtained by treating crude oil by standard purification, decoloration,and/or deodorizing processes. The microbial oil further contains “finaloil”, which is refined oil diluted with vegetable oil. The“microorganisms” are not limited, but include classificationstaxonomically associated with any “microalgae”, “labyrinthulids”, andthe deposited microorganisms described in the present specification. Theterms “labyrinthulid”, “genus Parietichytrium”, “genus Schizochytrium”,and “genus Thraustochytrium” when used in association with any microbialoils of a deposited microorganism described herein are based on currenttaxonomic classification including phylogenetically usable information,and are not intended to be limiting if the taxonomic classification isrevised after the filing date of the present application. Lipids

In the present invention, lipids are lipids produced by labyrinthulids,and are mainly triglycerides, diglycerides, monoglycerides,phospholipids, free fatty acids, sterols, carotenoids, hydrocarbons, andthe like.

[Lipids]

In the present invention, lipids are lipids produced by labyrinthulids,and are mainly triglycerides, diglycerides, monoglycerides,phospholipids, free fatty acids, sterols, carotenoids, hydrocarbons, andthe like.

[Polyunsaturated Fatty Acids]

In the present invention, a polyunsaturated fatty acid (PUFA) is a fattyacid having not less than 18 carbon atoms and not less than three doublebonds, and more preferably a fatty acid having not less than 20 carbonatoms and not less than three double bonds. Specific examples includelinoleic acid (LA, 18:2n-6), α-linolenic acid (ALA, 18:3n-3),γ-linolenic acid (GLA, 18:3n-6), stearidonic acid (STA, 18:4n-3),dihomo-γ-linolenic acid (DGLA, 20:3n-6), eicosatetraenoic acid (ETA,20:4n-3), arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA,20:5n-3), docosatetraenoic acid (DTA, 22:4n-6), n-3 docosapentaenoicacid (n-3 DPA, 22:5n-3), n-6 docosapentaenoic acid (n-6 DPA, 22:5n-6),and docosahexaenoic acid (DHA, 22:6n-3). In the present specification,arachidonic acid is also expressed as ARA. The total fatty acidcomposition means the composition of fatty acids detected after amicroorganism is cultured and freeze dried and the fatty acids are thenmethyl-esterified and analyzed by GC. Specifically, the total fatty acidcomposition is the composition of fatty acids having from 14 to 22carbon chains. In the present invention, GC area means the peak area ofthe GC chart. A proportion relative to the total fatty acid compositionmeans the proportion of the peak area of the targeting fatty acidrelative to the total of peak areas of the entire fatty acidcomposition, and is expressed as a percentage. In the present invention,C20 PUFA/C22 PUFA means the value obtained by dividing the total of GCpeak areas of polyunsaturated fatty acids having 20 carbon chains by thetotal of GC peak areas of polyunsaturated fatty acids having 22 carbonchains. In the present invention, n-6 PUFA/n-3 PUFA means the valueobtained by dividing the total of peak areas of the GC chart of ω6 fattyacids having not less than 20 carbon chains by the total of peak areasof the GC chart of 03 fatty acids having not less than 20 carbon chains.

[PUFA Biosynthesis Pathways]

Two different pathways associated with PUFA biosynthesis are known. Oneis a pathway that produces polyunsaturated fatty acids (PUFAs) usingpolyketide synthase (PKS). In the present invention, this metabolicpathway will be called the PUFA-PKS pathway or the PKS pathway. In thepresent invention, a PKS gene means a gene that encodes a protein thatconstitutes polyketide synthase. Polyketide synthase is an enzyme thatcatalyzes a reaction in which a long-chain substrate such as malonyl-CoAis condensed multiple times in a starter substrate such as acetyl-CoA.Polyketide synthase has generally been known as an enzyme involved inbiosynthesis of secondary metabolites of plants, fungi, and the like,but it has also been reported to be involved in PUFA biosynthesis incertain organisms. For example, the marine bacillus Shewanella produceseicosapentaenoic acid (EPA) using this enzyme (Non-patent Document 8).Polyketide synthase is known to be similarly involved in PUFAbiosynthesis in certain labyrinthulids as well.

The other is a pathway in which, starting from a fatty acid such aspalmitic acid, desaturation by desaturase and chain elongation byelongase are repeated to produce PUFAs such as EPA and DHA. In thepresent invention, this is called the elongase-desaturase pathway.Examples of the enzymes that constitute this system include fatty acidsynthesis associated enzymes such as C20 elongase and Δ4 desaturase.

[PUFA Biosynthesis Pathway Differentiation Method]

A method for differentiating between PUFA biosynthesis pathways oflabyrinthulids will be described below. However, this is only oneexample, and needless to say, differentiation does not have to beperformed by this method.

As stated above, two PUFA biosynthesis pathways in labyrinthulids areknown. One is the PUFA-PKS pathway, by which DHA and n-6 DPA areproduced. In this pathway, no PUFAs other than DHA and n-6 DPA aresubstantially produced such configuration differs greatly from theelongase-desaturase pathway, which is the other PUFA biosynthesispathway.

The other pathway, namely the elongase-desaturase pathway, isillustrated in FIG. 1. Among the enzymes constituting this pathway, Δ4desaturase is an enzyme involved in converting n-3 DPA to DHA. Thisenzyme is also involved in converting DTA to n-6 DPA. Thus, since theseconversions do not take place when the gene of this enzyme is disrupted,n-3 DPA and DTA, which are substrates of this enzyme, accumulate, andconversely, DHA and n-6 DPA, which are products of this enzyme,decrease. In a labyrinthulid in which this Δ4 desaturase gene has beendisrupted, in a case where the products DHA and/or n-6 DPA arecompletely undetected or markedly decreased, it is judged that thelabyrinthulid has very weak or no PUFA (particularly DHA and/or n-6 DPA)production activity via the endogenous PUFA-PKS pathway.

It is also possible to select C20 elongase rather than Δ4 desaturase anddisruption the gene of that enzyme instead. Specifically, C20 elongaseis an enzyme involved in converting EPA to n-3 DPA. This enzyme is alsoinvolved in converting ARA to DTA. Thus, since these conversions do nottake place when the gene of this enzyme is disrupted, EPA and ARA, whichare substrates of this enzyme, accumulate, and conversely, n-3 DPA andDTA, which are products of this enzyme, decrease. As a result, DHA andn-6 DPA, which have n-3 DPA and DTA as substrates, also decrease. In alabyrinthulid in which this C20 elongase gene has been disrupted, whenthe products DHA and/or n-6 DPA are completely undetected or markedlydecreased, it is judged that the labyrinthulid has very weak or no PUFA(particularly DHA and/or n-6 DPA) production activity via the endogenousPUFA-PKS pathway.

Gene disruption is performed by transforming something designed suchthat gene products do not produce activity using an antibioticresistance gene or the like in all or a portion of the target gene, intoa target cell by a method such as a gene gun, electroporation, or geneediting. Gene silencing is performed by transforming an antisense genedesigned such that expression of the target gene is inhibited or a genein which RNAi is expressed, into a cell by a method such as a gene gun,electroporation, or gene editing. Gene disruption and silencing are notlimited to these methods provided that expression of the target gene ishindered. When disruption or silencing is performed with a PKS gene asthe target, the target is not limited provided that enzyme activity canbe eliminated or inhibited, but OrfA is preferably used as the target.

The present invention provides labyrinthulids having very weak or noPUFA producing activity via the endogenous PUFA-PKS pathway and havingPUFA-producing activity via the endogenous elongase-desaturase pathway.

Furthermore, the present invention also includes changing the fatty acidcomposition produced by a labyrinthulid by manipulating the genes ofenzymes constituting the elongase-desaturase pathway of thelabyrinthulid. In particular, the fatty acid composition produced by alabyrinthulid can be modified by: (1) disruption and/or silencing of afatty acid elongase gene, (2) disruption and/or silencing of a fattyacid desaturase gene, (3) transfer of a fatty acid elongase gene, (4)transfer of a fatty acid desaturase gene, and (5) a combination of theabove. For example, when stearidonic acid (STA) is desired, a fatty acidelongase gene involved in conversion of stearidonic acid toeicosatetraenoic acid (ETA), specifically the C18 elongase gene, may bedisrupted and/or silenced. Furthermore, for example, wheneicosapentaenoic acid (EPA) is desired, a fatty acid elongase geneinvolved in conversion of eicosatetraenoic acid to docosapentaenoic acid(DPA), specifically the C20 elongase gene, may be disrupted and/orsilenced. As another example, when eicosapentaenoic acid is desired, afatty acid desaturase gene that converts arachidonic acid (ARA) toeicosapentaenoic acid, specifically the ω3 desaturase gene, may betransformed. For a labyrinthulid that produces via theelongase-desaturase pathway and PUFA-PKS pathway, the fatty acidcomposition produced by the labyrinthulid can be modified by: (1)disruption and/or silencing of a fatty acid elongase gene, (2)disruption and/or silencing of a fatty acid desaturase gene, (3)transfer of a fatty acid elongase gene. (4) transfer of a fatty aciddesaturase gene, and (5) a combination of the above, using amicroorganism in which the PUFA-PKS pathway has been disrupted orsilenced. For example, when stearidonic acid (STA) is desired, a fattyacid elongase gene involved in conversion of stearidonic acid toeicosatetraenoic acid (ETA), specifically the C18 elongase gene, may bedisrupted and/or silenced. Furthermore, for example, wheneicosapentaenoic acid (EPA) is desired, a fatty acid elongase geneinvolved in conversion of eicosatetraenoic acid to docosapentaenoic acid(DPA), specifically the C20 elongase gene, may be disrupted and/orsilenced. As another example, when eicosapentaenoic acid is desired, afatty acid desaturase gene that converts arachidonic acid (ARA) toeicosapentaenoic acid, specifically the ω3 desaturase gene, may betransformed.

A labyrinthulid (microorganism) in which the produced fatty acidcomposition has been modified is obtained by transformation of thelabyrinthulid. A labyrinthulid in which a fatty acid biosynthesisassociated gene has been transformed and/or disrupted can be used in,for example, production of unsaturated fatty acids. In production ofmunsaturated fatty acids, the conditions of other steps, productionequipment and instruments and the like are not particularly limitedprovided that the labyrinthulid that is used has very weak or no PUFAproduction activity via the endogenous PUFA-PKS pathway and has PUFAproduction activity via the endogenous elongase-desaturase pathway, oris a labyrinthulid in which the produced fatty acid composition has beenmodified as described above. Production of unsaturated fatty acidsincludes a step of culturing a labyrinthulid that has very weak or noPUFA production activity via the endogenous PUFA-PKS pathway and hasPUFA production activity via the endogenous elongase-desaturase pathway,or a labyrinthulid in which the produced fatty acid composition has beenmodified as described above. Unsaturated fatty acids are produced usingthese microorganisms and their culture medium.

The above cell culturing conditions (culture medium, culturetemperature, ventilation status, and the like) may be set as appropriateaccording to the type of cell, the targeted type and quantity ofunsaturated fatty acids, and the like. Furthermore, an unsaturated fattyacid in the present invention means a substance containing anunsaturated fatty acid, without limitation on its content, purity,shape, composition, and the like. In other words, in the presentinvention, cells in which the fatty acid composition has been modifiedor their culture media are themselves considered to be unsaturated fattyacids. Additionally, a step of purifying the unsaturated fatty acidsfrom these cells or culture media may be further included. As the methodof purifying the unsaturated fatty acids, a method known as apurification method for lipids (including complex lipids) such asunsaturated fatty acids may be applied.

[Method for Highly Accumulating Unsaturated Fatty Acids inLabyrinthulids]

Accumulation of unsaturated fatty acids in a labyrinthulid is achievedby culturing a labyrinthulid having very weak or no PUFA productionactivity via the endogenous PUFA-PKS pathway and having PUFA productionactivity via the endogenous elongase-desaturase pathway, or atransformant thereof. The labyrinthulid may be cultured in, for example,a solid culture medium, liquid culture medium, or the like. The culturemedium used at this time is not particularly limited provided that it isa medium commonly used for culturing labyrinthulids and appropriatelycombines, for example, glucose, fructose, saccharose, starch, glycerin,or the like as a carbon source, yeast extract, corn steep liquor,polypeptone, sodium glutamate, urea, ammonium acetate, ammonium sulfate,ammonium nitrate, ammonium chloride, sodium nitrate, or the like as anitrogen source, potassium phosphate or the like as an inorganic salt,and other necessary components. However, yeast extract-glucose medium(GY medium) is particularly preferably used. After preparation of theculture medium, the pH is adjusted to within the range of 3.0 to 8.0,and then the culture medium is sterilized by autoclave or the like.Culturing may be performed by aerated and agitated culturing, shakeculturing, or static culturing at 10 to 40° C., preferably 15 to 35° C.,for 1 to 14 days.

To collect the produced unsaturated fatty acids, the labyrinthulids aregrown in a culture medium and the microorganism cells obtained from thatculture medium are treated, the lipids (polyunsaturated fatty acids oroil- and fat-containing matter containing polyunsaturated fatty acids)inside the cells are released and collected. Specifically, lipidscontaining PUFAs can be obtained by collecting the labyrinthulidscultured in this manner by centrifugation or the like, performingtreatment such as drying and cell crushing as necessary, and performingextraction using an appropriate organic solvent or supercritical carbondioxide, liquefied dimethylether, or the like according to conventionalmethods.

The microbial oil obtained in the present invention is that whichsatisfies any of the following conditions. The microbial oil obtained inthe present invention contains ARA in a proportion of not less than 5%,not less than 7%, not less than 10%, or not less than 15% of the totalfatty acid composition. Oils and fats with high ARA obtained in thismanner may be used in applications such as nutritional supplements forinfants and health foods and medications for adults. ARA may be notgreater than 80%, not greater than 70%, not greater than 60%, or notgreater than 50% of the total fatty acid composition. The microbial oilobtained in the present invention contains DGLA in a proportion of notless than 2.5%, not less than 5%, or not less than 10% of the totalfatty acid composition. Microbial oil with high DGLA obtained in thismanner may be used in medicinal applications such as anti-inflammatoryagents. DGLA may be not greater than 80%, not greater than 70%, notgreater than 60%, or not greater than 50% of the total fatty acidcomposition. The microbial oil obtained in the present inventioncontains ETA in a proportion of not less than 0.35%, not less than 0.5%,not less than 0.75%, or not less than 1% of the total fatty acidcomposition. Microbial oil with high ETA obtained in this manner may beused in medicinal applications such as arthritis treatment. ETA may benot greater than 50%, not greater than 40%, not greater than 30%, or notgreater than 20% of the total fatty acid composition. The microbial oilobtained in the present invention contains EPA in a proportion of notless than 4%, not less than 6%, not less than 8%, not less than 10%, ornot less than 12% of the total fatty acid composition. Microbial oilwith high EPA obtained in this manner may be used in nutritionalsupplement applications and medicinal applications. EPA may be notgreater than 80%, not greater than 70%, not greater than 60%, or notgreater than 50% of the total fatty acid composition. The microbial oilobtained in the present invention contains n-6 DPA in a proportion ofnot greater than 0.20%, not greater than 0.15%, not greater than 0.1%,or not greater than 0.05% of the total fatty acid composition. Themicrobial oil with low n-6 DPA obtained in this manner does not tend tohinder the functions of other fatty acids. Furthermore, it isadvantageous in cases where removal of n-6 DPA by refinement is desired.n-6 DPA may be not less than 0.001%, not less than 0.005%, or not lessthan 0.01% of the total fatty acid composition. The microbial oilobtained in the present invention contains DHA in a proportion of notgreater than 0.50%, not greater than 0.3%, not greater than 0.2%, or notgreater than 0.1% of the total fatty acid composition. The microbial oilwith low DHA obtained in this manner does not tend to hinder thefunctions of other fatty acids. Furthermore, it is advantageous in caseswhere removal of DHA by refinement is desired. DHA may be not less than0.005%, not less than 0.01%, or not less than 0.05% of the total fattyacid composition. The microbial oil obtained in the present inventioncontains a total of DHA and n-6 DPA in a proportion of not greater than0.7%, not greater than 0.8%, not greater than 0.9%, or not greater than1.0% of the total fatty acid composition. The microbial oil with low DHAand n-6 DPA obtained in this manner does not tend to hinder thefunctions of other fatty acids and is stable against oxidation.Furthermore, it is advantageous in cases where removal of DHA and n-6DPA by refinement is desired. The total of DHA and n-6 DPA may be notless than 0.05%, not less than 0.1%, or not less than 0.5% of the totalfatty acid composition. The desired concentrations of each of thesefatty acids may be combined as desired, limited to a total of 100%.

In another aspect, the microbial oil obtained in the present inventionis that which satisfies any of the following conditions. In themicrobial oil obtained in the present invention, the value of LA/DHA byGC area is not less than 0.6, not less than 0.7, not less than 0.8, ornot less than 0.9, and not greater than 10, not greater than 9, notgreater than 8, not greater than 7, or not greater than 6. The microbialoil with a high LA/DHA value obtained in this manner is stable againstoxidation, and the functions of LA do not tend to be hindered by DHA. Inthe microbial oil obtained in the present invention, the value ofGLA/DHA by GC area is not less than 0.35, not less than 0.4, not lessthan 0.5, not less than 0.6, or not less than 0.7, and not greater than10, not greater than 9, not greater than 8, or not greater than 7. Themicrobial oil with a high GLA/DHA value obtained in this manner isstable against oxidation, and the functions of GLA do not tend to behindered by DHA.

In the microbial oil obtained in the present invention, the value ofDGLADHA by GC area is not less than 0.35, not less than 0.4, not lessthan 0.5, not less than 0.6, or not less than 0.7, and not greater than10, not greater than 9, not greater than 8, not greater than 7, or notgreater than 6. Microbial oil with a high DGLADHA value obtained in thismanner may be used in medications having an anti-inflammatory action. Inthe microbial oil obtained in the present invention, the value ofARA/DHA by GC area is not less than 0.7, not less than 0.8, not lessthan 0.9, or not less than 1.0, and not greater than 50, not greaterthan 45, not greater than 40, not greater than 35, or not greater than30. Microbial oil with a high ARADHA value obtained in this manner maybe used in modified milk for infants. The value of EPA/DHA by GC area isnot less than 0.35, not less than 0.4, not less than 0.5, not less than0.6, or not less than 0.7, and not greater than 50, not greater than 45,not greater than 40, not greater than 35, or not greater than 30. EPAused in typical medications and the like are mainly esters containingDHA, but the microbial oil with a high EPA/DHA value obtained in thismanner contains EPA with a high degree of purity, and can be used inhealth foods, medications, and the like.

The desired ratios of each of these fatty acids may be combined asdesired. In the microbial oil obtained in the present invention, thevalue of LA/EPA by GC area is not less than 0.06, not less than 0.07,not less than 0.08, or not less than 0.09, and not greater than 0.12,not greater than 0.1, not greater than 0.08, or not greater than 0.06.Microbial oil with a low LA/EPA value obtained in this manner may beused in infusion fluids with a low anti-inflammatory action and thelike. In the microbial oil obtained in the present invention, the valueof GLA/EPA by GC area is not less than 0.04 or not less than 0.45, andnot greater than 0.12, not greater than 0.1, or not greater than 0.8.Microbial oil with a low GLA/EPA value obtained in this manner may beused in infusion fluids with a low anti-inflammatory action and thelike. In the microbial oil obtained in the present invention, the valueof DTA/EPA by GC area is not less than 0.01, not less than 0.02, notless than 0.03, not less than 0.04, or not less than 0.05, and notgreater than 0.4, not greater than 0.35, or not greater than 0.3. Themicrobial oil with a low DTA/EPA value obtained in this manner is usefulfor separating high-purity EPA since DTA and EPA are close to each otherin GC. The desired ratios of each of these fatty acids may be combinedas desired.

In the microbial oil obtained in the present invention, the value ofDTA/ARA by GC area is not less than 0.01, not less than 0.03, not lessthan 0.05, not less than 0.07, or not less than 0.1, and not greaterthan 0.45, not greater than 0.4, not greater than 0.35, or not greaterthan 0.3. Microbial oil with a low DTA/ARA value obtained in this mannermay be used in formulated milk for infants. In the microbial oilobtained in the present invention, the value of DTA/DGLA by GC area isnot less than 0.01, not less than 0.05, not less than 0.1, not less than0.15, or not less than 0.2, and not greater than 1.45, not greater than1.4, not greater than 1.3, not greater than 1.2, or not greater than1.1. Microbial oil with a low DTA/DGLA value obtained in this manner maybe used in medications having an anti-inflammatory action and the like.In the microbial oil obtained in the present invention, the value ofLA/n-6 DPA by GC area is not less than 0.4, not less than 0.5, not lessthan 0.6, not less than 0.7, or not less than 0.8, and not greater than20, not greater than 18, not greater than 16, not greater than 14, ornot greater than 12. Microbial oil with a high LA/n-6 DPA value obtainedin this manner may be used in edible oils. In the microbial oil obtainedin the present invention, the value of GLA/n-6 DPA by GC area is notless than 0.2, not less than 0.4, not less than 0.6, or not less than0.8, and not greater than 10, not greater than 8, not greater than 6, ornot greater than 4. Microbial oil with a high GLA/n-6 DPA value obtainedin this manner may be used in health foods and supplements. In themicrobial oil obtained in the present invention, the value of DGLA/n-6DPA by GC area is not less than 0.35, not less than 0.5, not less than0.75, or not less than 1.0, and not greater than 30, not greater than27, not greater than 25, not greater than 22, or not greater than 20.Microbial oil with a high DGLA/n-6 DPA value obtained in this manner maybe used in medications having an anti-inflammatory action. In themicrobial oil obtained in the present invention, the value of ARA/n-6DPA by GC area is not less than 0.7, not less than 1.0, not less than2.0, or not less than 3.0, and not greater than 60, not greater than 50,not greater than 40, or not greater than 30. Microbial oil with a highLA/n-6 DPA value obtained in this manner may be used in formulated milkfor infants. In the microbial oil obtained in the present invention, thevalue of EPA/n-6 DPA by GC area is not less than 0.4, not less than 0.6,not less than 0.8, not less than 1, not less than 2, or not less than 5,and not greater than 70, not greater than 60, not greater than 50, notgreater than 40, or not greater than 30. Microbial oil with a highEPA/n-6 DPA value obtained in this manner may be used in health foods,supplements, and the like. The desired ratios of each of these fattyacids may be combined as desired.

In the microbial oil obtained in the present invention, the value ofDGLA/LA by GC area is not less than 1.4, not less than 2, or not lessthan 3, and not greater than 10, not greater than 9, not greater than 8,or not greater than 7. The microbial oil obtained in this manner may beused in foods and supplements having an anti-inflammatory action. In themicrobial oil obtained in the present invention, the value of ARA/LA byGC area is not less than 5.1, not less than 7, not less than 9, or notless than 11, and not greater than 20, not greater than 17, not greaterthan 15, or not greater than 12. Microbial oil with a high ARA/LA valueobtained in this manner may be used in animal feed and formulated milkfor infants. In the microbial oil obtained in the present invention, thevalue of EPA/LA by GC area is not less than 5.5, not less than 7, notless than 9, not less than 11, or not less than 13, and not greater than30, not greater than 25, not greater than 22, or not greater than 20.Microbial oil with a high EPA/LA value obtained in this manner may beused in medications and health foods. In the microbial oil obtained inthe present invention, the value of DTA/LA by GC area is not less than0.01, not less than 0.05, not less than 0.07, or not less than 0.1, andnot greater than 0.4, not greater than 0.35, not greater than 0.33, notgreater than 0.3, or not greater than 0.28. Microbial oil with a lowDTA/LA value obtained in this manner may be used in animal feed andfoods. The desired ratios of each of these fatty acids may be combinedas desired.

In the microbial oil obtained in the present invention, the value ofDGLA/GLA by GC area is not less than 4.5, not less than 5, not less than6, or not less than 7, and not greater than 20, not greater than 17, notgreater than 15, or not greater than 12. Microbial oil with a highDGLA/GLA value obtained in this manner may be used in medications havingan anti-inflammatory action and the like. In the microbial oil obtainedin the present invention, the value of ARA/GLA by GC area is not lessthan 9, not less than 10, not less than 12, not less than 13, or notless than 14, and not greater than 30, not greater than 28, not greaterthan 26, not greater than 24, or not greater than 22. Microbial oil witha high ARA/GLA value obtained in this manner may be used in formulatedmilk for infants. The desired ratios of each of these fatty acids may becombined as desired.

In the microbial oil obtained in the present invention, the value of n-6DPA/DTA by GC area is not less than 0.001, not less than 0.01, or notless than 0.02, and not greater than 1.5, not greater than 1.4, notgreater than 1.3, not greater than 1.2, or not greater than 1.1. Themicrobial oil obtained in this manner may be used in foods that preventarteriosclerosis and the like. In the microbial oil obtained in thepresent invention, the value of DHA/n-3 DPA by GC area is not less than0.001, not less than 0.01, or not less than 0.02, and not greater than4, not greater than 4.5, not greater than 4, or not greater than 3.5.The microbial oil obtained in this manner may be used in animal feed orhealth foods. In the microbial oil obtained in the present invention,the value of C20 PUFA/C22 PUFA by GC area is not less than 0.5, not lessthan 0.7, not less than 1, or not less than 1.2, and not greater than50, not greater than 45, not greater than 40, or not greater than 35.Microbial oil with a high C20 PUFA/C22 PUFA value obtained in thismanner may be used in health foods and supplements. In the microbial oilobtained in the present invention, the value of n-6 PUFA/n-3 PUFA by GCarea is not less than 1.8, not less than 2, not less than 2.5, or notless than 3, and not greater than 100, not greater than 80, not greaterthan 70, not greater than 60, or not greater than 50. Microbial oil witha high n-6 PUFA/n-3 PUFA value obtained in this manner may be used inedible oils and animal feed. The desired ratios of each of these fattyacids may be combined as desired.

The desired ratios of each of these fatty acids may be combined asdesired. The desired concentrations of each of these fatty acids may becombined as desired, limited to a total of 100%.

The unsaturated fatty acids of the present invention also includevarious medications, foods, animal feeds, and industrial products, andthe fields of use thereof are not particularly limited. The foodscontaining the unsaturated fatty acid-containing oils and fats of thepresent invention also include health foods such as supplements and foodadditives and the like. Examples of the industrial products includeanimal feed for organisms other than humans, films, biodegradableplastics, functional fibers, lubricating oils, and detergents.

Next, the present invention will be specifically described based onexamples. Furthermore, in the present specification, the features ofeach invention described in embodiments related to each aspect of theinvention may be combined as desired to form new embodiments, and it isto be understood that such new embodiments may be included in each ofthe aspects of the present invention.

Example 1 [Labyrinthulids and Culturing Method/Storage Method Thereof](1) Strains Used in the Present Invention

Parietichytrium sp. SEK358 (FERM BP-11405), Parietichytrium sarkarianumSEK364 (FERM BP-11298), and Parietichytrium sp. SEK571 (FERM BP-11406)were shared from the Department of Engineering at Konan University.Thraustochytrium aureum ATCC 34304 was shared from ATCC.

(2) Culture Medium Composition

i. Agar Plate Culture Medium Composition

PDA Agar Plate Culture Medium

0.78% (w/v) of potato dextrose agar medium (available from NissuiPharmaceutical Co., Ltd.), 1.75% (w/v) of Sealife (available fromMarintec Co., Ltd.), and 1.21% (w/v) of agar powder (available fromNacalai Tesque, Inc.) were mixed and then sterilized by autoclave for 20min at 121° C. After sufficient cooling, ampicillin sodium salt(available from Nacalai Tesque, Inc.) was added so as to result in afinal concentration of 100 μg/mL. This was dispensed into a Petri dishand left to stand at a level location to solidify.

ii. Liquid Culture Medium Composition

GY Liquid Culture Medium

3.18% (w/v) of glucose (available from Nacalai Tesque, Inc.), 1.06%(w/v) of dry yeast extract (available from Nacalai Tesque, Inc.), and1.75% (w/v) of Sealife (available from Marintec Co., Ltd.) were mixedand then sterilized by autoclave for 20 min at 121° C. Ampicillin sodiumsalt (available from Nacalai Tesque, Inc.) was then added so as toresult in a final concentration of 100 μg/mL.

PD Liquid Culture Medium

0.48% (w/v) of potato dextrose (available from Difco Laboratories Inc.)and 1.75% (w/v) of Sealife (available from Marintec Co., Ltd.) weremixed and then sterilized by autoclave for 20 min at 121° C. Ampicillinsodium salt (available from Nacalai Tesque, Inc.) was then added so asto result in a final concentration of 100 μg/mL.

(3) Culturing Method

i. Agar Plate Culturing

Labyrinthulea cells were inoculated using a platinum loop or a spreaderand then static cultured at 25° C., thereby causing emergence ofcolonies. Subculturing was performed by extracting colonies using aplatinum loop and suspending them in sterilized physiological salinesolution, and then spreading this suspension using a platinum loop or aspreader. Furthermore, as necessary, it was transformed to a liquidculture by inoculating cells in a liquid culture medium on a flat plate.

ii. Liquid Culturing

Labyrinthulea cells were inoculated, and suspension culturing wasperformed with stirring at 150 rpm at 25° C. using an Erlenmeyer flaskor a test tube. Subculturing was performed by adding a culture solutionin which growth was confirmed from the logarithmic growth phase to thestationary phase, in a volume ratio of 1/200 to 1/10 to a fresh GY or PDliquid culture medium. Furthermore, as necessary, it was transformed toan agar plate culture by spreading the cell culture solution on a PDAagar plate culture medium.

(4) Preservation/Storage Method of Labyrinthulids

In addition to subculturing, cryopreservation was performed by producingglycerol stock. Specifically, glycerol (available from Nacalai Tesque,Inc.) was added to a cell suspension that used a GY liquid culturemedium from the logarithmic growth phase to the stationary phase, so asto result in a final concentration of 15% (v/v), and this was stored ina deep freezer at −80° C.

Example 2 Measurement of Fatty Acid Composition of Lipids Produced byC20 Elongase Gene Disruption and Transformation Strain ofParietichytrium Sarkarianum SEK364 [Example 2-1]: Subcloning of SV40Terminator Sequence

An SV40 terminator sequence was amplified with PrimeSTAR HS DNAPolymerase (available from Takara Bio Inc.) using a pcDNA 3.1 myc-Hisvector (available from Invitrogen Corp.) as a template. The PCR primersused were as shown below. RHO58 was set on the SV40 terminator sequence,and includes BglII and BamHI linker sequences. RHO52 was set on the SV40terminator sequence, and includes a BglII sequence. [RHO58: 34mer:5′-CAG ATC TGG ATC CGC GAA ATG ACC GAC CAA GCG A-3′ (SEQ ID NO: 1),RHO52: 24mer: 5′-ACG CAA TTA ATG TGA GAT CTA GCT-3′ (SEQ ID NO: 2)].After amplification under the following conditions, it was cloned inpGEM-T Easy Vector (available from Promega Corporation). [PCR cycles:98° C. 2 min/98° C. 30 sec, 60° C. 30 sec, 72° C. 1 min, 30 cycles/72°C. 1 min]. After amplification with E. coli, the sequence was confirmedusing a Dye Terminator Cycle Sequencing Kit (available from BeckmanCoulter Inc.). This was named pRH27.

The plasmid (pRH27) containing the subcloned SV40 terminator sequence(342 bp, SEQ ID NO: 3) is illustrated in FIG. 2.

[Example 2-2]: Production of Artificially Synthesized NeomycinResistance Gene Cassette

Thraustochytrium aureum ATCC 34304 strain was cultured in a GY culturemedium, and cells of the latter logarithmic growth phase werecentrifuged for 5 min at 4° C. at 3500×g to form pellets, and thepellets were frozen with liquid nitrogen and then crushed. After phenolextraction of the crushed cell liquid, ethanol precipitation wasperformed and the precipitate was dissolved in a TE solution. Thenucleic acids dissolved in the TE solution were treated with RNase for30 min at 37° C., and after further phenol extraction, ethanolprecipitation was performed and the precipitate was dissolved in a TEsolution. A260/280 was measured and the DNA concentration wascalculated.

Using this as a template, an ubiquitin promoter sequence (619 bp, SEQ IDNO: 4) was amplified with PrimeSTAR HS DNA Polymerase with GC Buffer(available from Takara Bio Inc.). The PCR primers used were as shownbelow. RHO53 was set on the ubiquitin promoter sequence, and includes aBglII linker sequence. TKO1 includes the ubiquitin promoter sequence andan artificially synthesized neomycin resistance gene sequence. [RHO53:36mer: 5′-CCC AGA TCT GCC GCA GCG CCT GGT GCA CCC GCC GGG-3′ (SEQ ID NO:5), TKO1: 58mer: 5′-CGT GAA GGC CGT CCT GTT CAA TCA TGT TGG CTA GTG TTGCTT AGG TCG CTT GCT GCT G-3′ (SEQ ID NO: 6)]. [PCR cycles: 98° C. 2min/98° C. 10 sec, 68° 1 min, 30 cycles/68° C. 1 min].

Using the artificially synthesized neomycin resistance gene sequence asa template, an artificially synthesized neomycin resistance genesequence (826 bp, SEQ ID NO: 7) was amplified with PrimeSTAR HS DNAPolymerase with GC Buffer (available from Takara Bio Inc.). The PCRprimers used were as shown below. TKO2 includes the ubiquitin promotersequence and the artificially synthesized neomycin resistance genesequence. RHO57 includes the artificially synthesized neomycinresistance gene sequence and has a BglII linker sequence. [TKO2: 54mer:5′-AGC GAC CTA AGC AAC ACT AGC CAA CAT GAT TGA ACA GGA CGG CCT TCA CGCTGG-3′ (SEQ ID NO: 8), RHOS57: 26mer: 5′-CAG ATC TCA AAA GAA CTC GTC CAGGA-3′ (SEQ ID NO: 9)] [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° 1min, 30 cycles/68° C. 1 min].

Using SEQ ID NO: 4 and SEQ ID NO: 7 as templates, fusion PCR wasperformed using RHO53 (SEQ ID NO: 5) and RHO57 (SEQ ID NO: 9) accordingto the method described in Non-patent Document 9. Amplification wasperformed using LA Taq Hot Start Version (available from Takara BioInc.) as the enzyme with PCR cycles under conditions below: 94° C. 2min/94° C. 20 sec, 55° C. 30 sec, 68° C. 1 min, 30 cycles/68° C. 1 min(1° C./10 sec from 55° C. to 68° C.), and then the amplified product wasdigested with BglII. (FIG. 3).

The Thraustochytrium aureum ATCC 34304-derived ubiquitinpromoter—artificially synthesized neomycin resistance gene sequence(1395 bp, SEQ ID NO: 10) fused as described above was digested withBglII, and the resultant was bound to the BamHI site of pRH27 describedin Example 2-1. After amplification of the produced plasmid with E.coli, the sequence was confirmed using a Dye Terminator Cycle SequencingKit (available from Beckman Coulter Inc.). This was named pRH31.

The produced artificially synthesized neomycin resistance gene cassette(pRH31) is illustrated in FIG. 4.

[Example 2-3]: Production of Hygromycin Resistance Gene Cassette

Using Thraustochytrium aureum ATCC 34304 genome DNA as a template, anubiquitin promoter sequence (617 bp, SEQ ID NO: 11) was amplified withPrimeSTAR HS DNA Polymerase with GC Buffer (available from Takara BioInc.). The PCR primers used were as shown below. RHO53 was set on theubiquitin promoter sequence, and includes a BglII linker sequence. KSO8includes the ubiquitin promoter sequence and a hygromycin resistancegene sequence. [RHO53: 36mer: 5′-CCC AGA TCT GCC GCA GCG CCT GGT GCA CCCGCC GGG-3′ (described in Example 2-2, SEQ ID NO: 5), KSO8: 58mer: 5′-TCGCGG TGA GTT CAG GCT TTT TCA TGT TGG CTA GTG TTG CTT AGG TCG CTT GCT GCTG-3′ (SEQ ID NO: 12)] [PCR cycles: 98° C. 2 min/98° C. 30 see, 68° 2min, 30 cycles/68° C. 2 min]

Using pcDNA 3.1/Hygro (available from Invitrogen Corp.) as a template, ahygromycin resistance gene (1058 bp, SEQ ID NO: 13) was amplified withPrimeSTAR HS DNA Polymerase with GC Buffer (available from Takara BioInc.). The PCR primers used were as shown below. KSO7 includes theubiquitin promoter sequence and the hygromycin resistance gene sequence.RHOS6 includes the hygromycin resistance gene sequence and has a BglIIlinker sequence. [KSO7: 56mer: 5′-AGC GAC CTA AGC AAC ACT AGC CAA CATGAA AAA GCC TGA ACT CAC CGC GAC GTC TG-3′ (SEQ ID NO: 14), RHO56: 36mer:5′-CAG ATC TCT ATT CCT TTG CCC TCG GAC GAG TGC TGG-3′ (SEQ ID NO: 15)].[PCR cycles: 98° C. 2 min/98° C. 30 sec, 68° 2 min, 30 cycles/68° C. 2min]

Using SEQ ID NO: 11 and SEQ ID NO: 13 as templates, fusion PCR wasperformed using RHO53 (described in Example 2-2. SEQ ID NO: 5) and RHO56(SEQ ID NO: 15) according to the method described in Non-patent Document9. Amplification was performed using LA Taq Hot Start Version (availablefrom Takara Bio Inc.) as the enzyme under the following conditions, andthen the amplified product was digested with BglII. [PCR cycles: 94° C.2 min/94° C. 20 see, 55° C. 30 sec, 68° C. 1 min, 30 cycles/68° C. 1 min(1° C./10 sec from 55° C. to 68° C.)] (FIG. 5).

The Thraustochytrium aureum ATCC 34304-derived ubiquitin promoter—pcDNA3.1/Hygro (available from Invitrogen Corp.)-derived hygromycinresistance gene (1625 bp, SEQ ID NO: 16) fused as described above wasdigested with BglII, and the resultant was bound to the BamHI site ofpRH27 described in Example 2-1, FIG. 2. After amplification of theproduced plasmid with E. coli, the sequence was confirmed using a DyeTerminator Cycle Sequencing Kit (available from Beckman Coulter Inc.).This was named pRH32.

The produced hygromycin resistance gene cassette (pRH32) is illustratedin FIG. 6.

[Example 2-4]: Cloning of Genus Parietichytrium C20 Elongase Gene

Genome DNA of Parietichytrium sarkarianum SEK364 was extracted by themethod described in Example 2-2, and the genome was read.

With the region conserved in the C20 elongase gene as a target, aforward oligonucleotide (PsTaELO2 F1; 5′-CCT TCG GCG CTC CTC TTA TGT ATGT-3′) (SEQ ID NO: 17) and a reverse oligonucleotide (PsTaELO2 R2; 5′-CAATGC AAG AGG CGA ACT GGG AGA G-3′) (SEQ ID NO: 18) were synthesized.Next, using the Parietichytrium sarkarianum SEK364 genome DNA preparedby the method described in Example 2-2 as a template, PCR was performedusing the oligonucleotides PsTaELO2 F1 and PsTaELO2 R2 using LA Taq HotStart Version (available from Takara Bio Inc.). [PCR cycles: 98° C. 1min/98° C. 10 see, 60° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 7min/4° C. r]. The obtained specific amplification product underwent gelpurification, and when the base sequence thereof was analyzed by directsequencing, it exhibited significant homology to a known C20 elongasegene sequence. This shows that it was a partial sequence of the C20elongase gene derived from Parietichytrium sarkarianum SEK364.

Then, the C20 elongase gene derived from Parietichytrium sarkarianumSEK364 was cloned by 3′- and 5′-RACE in the same manner as ComparativeExample 1-2 to be described later. First, forward oligonucleotideprimers (PsRACE F1; 5′-TGG GGC TCT GGA ACC GCT GCT TAC G-3′) (SEQ ID NO:19) and (PsRACE F2; 5′-CTT CCA GCT CTC CCA GTT CGC CTC T-3′) (SEQ ID NO:20), and reverse oligonucleotide primers (PsRACE R1; 5′-CGG GTT GTT GATGTT GAG CGA GGT G-3′) (SEQ ID NO: 21) and (PsRACE R2: 5′-CCC ACG CCA TCCACG AGC ACA CCA C-3′) (SEQ ID NO: 22) were designed. Next, using a cDNAlibrary produced by SMART RACE cDNA Amplification Kit (trade name;available from Clontech Laboratories, Inc.) as a template, 3′- and5′-RACE were performed using a synthetic adapter-specificoligonucleotide and the above oligonucleotide PsRACE F1 or PsRACE R1.[PCR cycles: 94° C. 30 sec 5 cycles/94° C. 30 sec, 70° C. 30 sec, 72° C.3 min, 5 cycles/94° C. 30 sec, 68° C. 30 sec, 72° C. 3 min, 25 cycles/4°C. ∞]. Then, using the two obtained RACE products as templates, nestedPCR was performed using a synthetic adapter-specific oligonucleotide andthe above oligonucleotide PsRACE F2 or PsRACE R2. [PCR cycles: 94° C. 1min/94° C. 30 sec, 68° C. 30 sec, 72° C. 3 min, 25 cycles/72° C. 10min/4° C. ∞]. The obtained specific amplification product underwent gelpurification, and when the base sequence thereof was analyzed after TAcloning using pGEM-Easy Vector (available from Promega Corporation), itwas confirmed to be the C20 elongase gene derived from Parietichytriumsarkarianum SEK364.

Additionally, using the genus Parietichytrium genome DNA extracted bythe method described in Example 2-2 as a template, a sequence containinga C20 elongase gene sequence (957 bp. SEQ ID NO: 23) was amplified withLA Taq Hot Start Version (available from Takara Bio Inc.). The PCRprimers used were as shown below. RHO153 includes a start codon, and hasa BamHI site as a linker sequence. RHO154 includes a stop codon, and hasa BamHI site as a linker sequence. [RHO153: 32mer: 5′-CCC GGA TCC ATGGCA GCT CGC GTG GAG AAA CA-3′ (SEQ ID NO: 24), RHO154: 33mer: 5′-CCC GGATCC TTA CTG AGC CTT CTT GGA GGT CTC-3′ (SEQ ID NO: 25)]. [PCR cycles:98° C. 2 min/98° C. 10 sec, 68° 1 min, 30 cycles/68° C. 2 min].

The obtained DNA fragment was cloned in pGEM-T Easy Vector, and afteramplification with E. coli, the sequence was confirmed using a DyeTerminator Cycle Sequencing Kit (available from Beckman Coulter Inc.).

The genus Parietichytrium C20 elongase gene (936 bp, SEQ ID NO: 26) wascloned. This was named pRH80 (FIG. 7). The amino acid sequence is shownSEQ ID NO: 27.

[Example 2-5]: Production of Base Plasmid for Production of GenusParietichytrium C20 Elongase Gene Targeting Vector

A primer set that was set up in the reverse direction so as to insert aBglII site into the core portion of the C20 elongase gene sequence wasprepared using pRH80 (FIG. 7) produced in Example 2-4 as a template, andthe resultant was amplified with PrimeSTAR Max DNA Polymerase (availablefrom Takara Bio Inc.). The PCR primers used were as shown below and havea BglII linker sequence. [RHO155: 26mer: 5′-ACA AAG ATC TCG ACT GGA CCGACA CC-3′ (SEQ ID NO: 28), RHO156: 27mer: 5′-AGT CGA GAT CTT TGT CAG GAGGTG GAC-3′ (SEQ ID NO: 29)]. [PCR cycles: 98° C. 2 min/98° C. 10 sec,56° C. 15 sec, 72° C. 1 min, 30 cycles/72° C. 1 min]. Afteramplification under the above conditions, it was digested with BglII andthen self-ligated. After the ligated sample was amplified with E. coli,the sequence was confirmed using a Dye Terminator Cycle Sequencing Kit(available from Beckman Coulter Inc.). This was named pRH83. The C20elongase gene sequence (935 bp) in which a BglII site was inserted isshown in SEQ ID NO: 30.

FIG. 8 illustrates the produced base plasmid (pRH83) for producing agenus Parietichytrium C20 elongase gene targeting vector.

[Example 2-6]: Production of Targeting Vectors (Artificially SynthesizedNeomycin Gene and Hygromycin Resistance Gene)

pRH31 (FIG. 4) described in Example 2-2 was digested with BglII, and aDNA fragment containing an artificially synthesized neomycin resistancegene cassette was bound to the BglII site of pRH83 (FIG. 8) described inExample 2-5. This was named pRH85.

pRH32 (FIG. 6) described in Example 2-3 was digested with BglII, and aDNA fragment containing a hygromycin resistance gene cassette was boundto the BglII site of pRH83 (FIG. 8) described in Example 2-5. This wasnamed pRH86.

The two produced targeting vectors (pRH85 and 86) are illustrated inFIG. 9.

[Example 2-7]: C20 Elongase Gene Targeting Vector Transfer

Using the two targeting vectors produced in Example 2-6 as templates,the genes were amplified with PrimeSTAR Max DNA Polymerase (availablefrom Takara Bio Inc.) using RHO153 (described in Example 2-4. SEQ ID NO:24) and RHO154 (described in Example 2-4, SEQ ID NO: 25) as primers.[PCR cycles: 98° C. 2 min/98° C. 30 sec, 68° 2 min, 30 cycles/68° C. 2min]. After phenol chloroform extraction and chloroform extraction, theDNA underwent ethanol precipitation, and the precipitate was dissolvedin 0.1×TE. A260/280 was measured and the DNA concentration wascalculated. The transfer fragment obtained when pRH85 (FIG. 9) describedin Example 2-6 was used as a template was 2661 bp, and resulted in asequence composed of genus Parietichytrium C20 elongase gene fronthalf—SV40 terminator sequence—artificially synthesized neomycinresistance gene sequence—ubiquitin promoter sequence—genusParietichytrium C20 elongase gene back half (SEQ ID NO: 31). Thetransfer fragment obtained when pRH86 (FIG. 9) described in Example 2-6was used as a template was 2892 bp, and resulted in a sequence composedof genus Parietichytrium C20 elongase gene front half—SV40 terminatorsequence—hygromycin resistance gene sequence—ubiquitin promotersequence—genus Parietichytrium C20 elongase gene back half (SEQ ID NO:32).

The Parietichytrium sarkarianum SEK364 strain was cultured for 4 days ina GY culture medium, and cells in the logarithmic growth phase were usedfor gene transfer. To cells corresponding to OD600=1 to 1.5, 0.625 μg ofDNA fragment was transformed by the gene gun method (microcarrier: 0.6micron gold particles, target distance: 6 cm, chamber vacuum: 26 mmHg,rupture disk: 1550 psi). After a recovery time of 24 hr, the transgeniccells were spread on a PDA agar plate culture medium (containing 2 mg/mLof G418 or containing 2 mg/mL of hygromycin). As a result, from 10 to 20cells of drug resistant strain per shot were obtained.

[Example 2-8]: Identification of C20 Elongase Gene Targeting HomologousRecombinant

Genome DNA of the Parietichytrium sarkarianum SEK364 strain, a C20elongase gene hetero homologous recombinant, and a C20 elongase genehomo homologous recombinant (gene disruption strain) were extracted bythe method described in Example 2-2, and then A260/280 was measured andthe DNA concentration was calculated. Using the genome DNA as templates,PCR for genome structure confirmation was performed using LA Taq HotStart Version (available from Takara Bio Inc.). The positions of theprimers used, the combinations used in amplification, and the expectedsizes of the amplification products are illustrated in FIG. 10. RHO184was set upstream of C20 elongase; RHO185 was set downstream: RHO142 andRHO143 were set on the artificially synthesized neomycin resistancegene; and RHO140 and RHO141 were set on the hygromycin resistance gene.[RHO140: 20mer: 5′-GGT TGA CGG CAA TTT CGA TG-3′ (SEQ ID NO: 33),RHO141: 22mer: 5′-CCT CCT ACA TCG AAG CTG AAA G-3′ (SEQ ID NO: 34),RHO142: 21mer: 5′-CTT CTC GGG CTT TAT CGA CTG-3′ (SEQ ID NO: 35),RHO143: 22mer: 5′-TAA GGT CGG TCT TGA CAA ACA G-3′ (SEQ ID NO: 36).RHO184: 24mer: 5′-AGT AGT CCC CGA TTT GGT AGT TGA-3′ (SEQ ID NO: 37),RHO185: 22mer: 5′-GGC AGA GAG CAA AAA CAC GAG C-3′ (SEQ ID NO: 38)].[PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° 4 min, 30 cycles/68° C. 7min].

A C20 elongase gene disruption strain in which there is no amplificationin the wild-type allele (Wt allele) and there is amplification in theartificially synthesized neomycin resistance gene allele (NeoR allele)and the hygromycin resistance gene allele (HygR allele) was obtained(FIG. 11).

Example 2-9: Change in Fatty Acid Composition by C20 Elongase GeneDisruption

The Parietichytrium sarkarianum SEK364 wild-type strain and the genedisruption strain thereof (C20 elongase gene disruption strain, C20 −/−)were cultured in a GY culture medium. The cells of the latter phase ofthe logarithmic growth phase were centrifuged for 10 min at 4° C. at3000 rpm to form pellets, and the obtained pellets were suspended in0.9% NaCl and washed. Then, the resultant was centrifuged for 10 min at4° C. at 3000 rpm, and the pellets were suspended in sterilized waterand washed. The resultant was further centrifuged for 10 min at 3000rpm, the supernatant was removed and the precipitate was freeze-dried.

To the freeze-dried cells, 2 mL of methanolic KOH (7.5% KOH in 95%methanol) was added, and after vortexing, the cells were crushed byultrasound (80° C., 30 min). Then, 500 μL of sterilized water was addedand vortexing was performed, and then 2 mL of n-hexane was added andvortexing was performed. The resultant was then centrifuged for 10 minat 3000 rpm, and the top layer was discarded. Another 2 mL of n-hexanewas added and vortexing was performed. The resultant was centrifuged for10 min at 3000 rpm, and the top layer was discarded. One mL of 6 N HClwas added to the remaining bottom layer and vortexing was performed, andthen 2 mL of n-hexane was added and vortexing was performed. Theresultant was then centrifuged for 10 min at 3000 rpm, and the top layerwas collected. Another 2 mL of n-hexane was added and vortexing wasperformed. The resultant was centrifuged for 10 min at 3000 rpm, and thetop layer was collected. The collected top layer was concentrated anddried with nitrogen gas. Two mL of 3 N methanolic HCl was added to theconcentrated and dried sample, and the resultant was incubated overnightat 80° C.

The sample was cooled to room temperature, and 1 mL of 0.9% NaCl wasadded. Then, 2 mL of n-hexane was added and vortexing was performed. Theresultant was centrifuged for 10 min at 3000 rpm, and the top layer wascollected. Another 2 mL of n-hexane was added and vortexing wasperformed. The resultant was centrifuged for 10 min at 3000 rpm, and thetop layer was collected. A small amount of anhydrous sodium sulfate wasadded to the collected top layer and then vortexing was performed. Theresultant was centrifuged for 10 min at 3000 rpm, and the top layer wascollected. The collected top layer was concentrated and dried withnitrogen gas. The concentrated and dried sample was dissolved in 0.5 mLof n-hexane, and 1 μL of the resultant was subjected to GC analysis. InGC analysis, measurement was performed using a gas chromatograph GC-2014(available from Shimadzu Corporation) under the following conditions.Column: HR-SS-10 (30 m×0.25 mm; available from Shinwa ChemicalIndustries Ltd.): column temperature: 150° C.→(5° C./min)→220° C. (10min); carrier gas: He (1.3 mL/min).

As a result, when the C20 elongase gene was disrupted in Parietichytriumsarkarianum SEK364, fatty acids having not less than 22 carbon chainsdecreased while fatty acids having 20 carbon chains increased (FIG. 12).FIG. 13 shows the proportion when the wild-type strain is taken as 100%.FIG. 13 shows that, of the total fatty acid composition, ARA is 25.22%,DGLA is 8.62%, ETA is 0.56%, EPA is 11.58%, n-6 DPA is 1.64%, and DHA is1.28%. FIG. 13 shows that, by GC area, LA/DHA is 5.8, GLA/DHA is 1.5,DGLA/DHA is 6.7, ARA/DHA is 19.7, EPA/DHA is 9.0, LA/EPA is 0.64,GLA/EPA is 0.16, DTA/EPA is 0.06, DTA/ARA is 0.03. DTA/DGLA is 0.08,LA/n-6 DPA is 4.5, GLA/n-6 DPA is 1.2, DGLA/n-6 DPA is 5.3, ARA/n-6 DPAis 15.4, EPA/n-6 DPA is 7.1, DGLA/LA is 1.2, ARA/LA is 3.4, EPA/LA is1.6, DTA/LA is 0.09, DGLA/GLA is 4.5. ARA/GLA is 13.2, n-6 DPA/DTA is2.4, DHA/n-3 DPA is 4.9, C20 PUFA/C22 PUFA is 11.94, and n-6 PUFA/n-3PUFA is 2.67.

In these results, arachidonic acid increased approximately 10-fold, EPAapproximately 8-fold, and DGLA approximately 16-fold, while DPAdecreased to approximately ¼ and DHA to approximately ⅕.

By selecting the labyrinthulid Parietichytrium sarkarianum SEK364 havingno PUFA-PKS pathway in this manner, a strain that accumulates PUFAsother than DHA and n-6 DPA can be produced without PUFA-PKS pathway genedisruption. This strain may also be used as a strain that produces EPAand/or ARA, and further disruption or transforming elongase ordesaturase genes can create strains that produce desired PUFAs.

Example 3 Measurement of Fatty Acid Composition of Lipids Produced by Δ4Desaturase Gene Disruption and Transformation Strain of Parietichytriumsarkarianum SEK364 [Example 3-1]: Cloning of Genus Parietichytrium Δ4Desaturase Gene

Genome DNA of Parietichytrium sp. SEK571 was extracted by the methoddescribed in Example 2-2. Using the extracted genome DNA as a template,a sequence containing the Δ4 desaturase gene sequence (5003 bp, SEQ IDNO: 39) was amplified with LA Taq Hot Start Version (available fromTakara Bio Inc.). The PCR primers used were as shown below. [RHO241:23mer: 5′-GTT TGA GGA GCG AGG CAT TTC TT-3′ (SEQ ID NO: 40), RHO242:23mer: 5′-AGT GCT CGT ACA ATG ACT GGC GT-3′ (SEQ ID NO: 41)].

The obtained DNA fragment was cloned in pGEM-T Easy Vector, and afteramplification with E. coli, the sequence was confirmed using a DyeTerminator Cycle Sequencing Kit (available from Beckman Coulter Inc.).This was named pRH112 (SEQ ID NO: 42).

The plasmid (pRH112) containing the genus of Parietichytrium Δ4desaturase gene sequence (1542 bp, SEQ ID NO: 43) is illustrated in FIG.14.

[Example 3-2]: Production of Plasmid Serving as Base for Production ofΔ4 Desaturase Gene Targeting Vector

Using pRH112 (FIG. 14) produced in Example 3-1 as a template, a primerset designed so as to delete the Δ4 desaturase gene and 600 bpdownstream of the Δ4 desaturase gene and to produce a BglII site in thedeleted portion was prepared. [RHO243: 26mer: 5′-GGC AAG ATC TAA CTT TCTGAG GCT CT-3′ (SEQ ID NO: 44), RHO244: 26mer: 5′-AAG TTA GAT CTT GCC TATTCC ACG AT-3′ (SEQ ID NO: 45)]. PrimeSTAR Max DNA Polymerase (availablefrom Takara Bio Inc.) was used in amplification. After the amplifiedsample was digested with BglII, the resultant was self-ligated. Afterthe ligated sample was amplified with E. coli, the sequence wasconfirmed using a Dye Terminator Cycle Sequencing Kit (available fromBeckman Coulter Inc.). This was named pRH117.

The produced plasmid (pRH117) serving as a base for production of a Δ4desaturase gene targeting vector is illustrated in FIG. 15.

[Example 3-3]: Production of Δ4 Desaturase Gene Targeting Vector

pRH31 (FIG. 4) described in Example 2-2 was digested with BgII, and aDNA fragment containing an artificially synthesized neomycin resistancegene cassette was bound to the BglII site of pRH17 (FIG. 15) describedin Example 3-2. This was named pRHI24 (FIG. 16). Using this pRHI24 as atemplate, a primer set that was set up so as to delete PstI wasprepared. [RHO261: 26mer: 5′-GTG CAG ACG CAG AAG AAG ACT GAC AA-3′ (SEQID NO: 46), RHO262: 25mer: 5′-CTT CTG CGT CTG CAC GAG GAA TCG A-3′ (SEQID NO: 47)]. PrimeSTAR Max DNA Polymerase (available from Takara BioInc.) was used in amplification. After transforming the PCR product intoE. coli and amplifying, the sequence was confirmed using a DyeTerminator Cycle Sequencing Kit (available from Beckman Coulter Inc.).This was named pRH126 (SEQ ID NO: 48).

The produced Δ4 desaturase gene targeting vector (pRHI26) is illustratedin FIG. 17.

[Example 3-4]: Transfer of Δ4 Desaturase Gene Targeting Vector toParietichytrium sarkarianum SEK364

Using the targeting vector pRHI26 (FIG. 17) produced in Example 3-3 as atemplate, the gene was amplified with PrimeSTAR Max DNA Polymerase(available from Takara Bio Inc.) using RHO241 (described in Example 3-1,SEQ ID NO: 40) and RHO242 (described in Example 3-1, SEQ ID NO: 41) asprimers. After phenol chloroform extraction and chloroform extraction,the DNA underwent ethanol precipitation, and the precipitate wasdissolved in 0.1 TE. A260/280 was measured and the DNA concentration wascalculated. The transfer fragment obtained when pRHI26 (FIG. 17)described in Example 3-3 was used as a template was 4562 bp.

The Parietichytrium sarkarianum SEK364 strain was cultured for 1 to 2days in a GY culture medium, and cells in the logarithmic growth phasewere used for gene transfer. To cells corresponding to OD600=1 to 2,0.625 μg of DNA fragment was transformed by the gene gun method(microcarrier: 0.6 micron gold particles, target distance: 6 cm, chambervacuum: 26 mmHg, rupture disk: 1550 psi). After a recovery time of 24hr, the transgenic cells were spread on a PDA agar plate culture mediumcontaining 1 mg/mL of G418. As a result, from 0 to 2 cells of drugresistant strain per shot were obtained.

[Example 3-5]: Identification of Δ4 Desaturase Gene Targeting HomologousRecombinant

Genome DNA of the Parietichytrium sarkarianum SEK364 strain and the Δ4desaturase gene disruption strain were extracted by the method describedin Example 2-2, and then A260/280 was measured and the DNA concentrationwas calculated. Using the genome DNA as templates, PCR for genomestructure confirmation was performed using PrimeSTAR GXL DNA Polymerase(available from Takara Bio Inc.). The positions of the primers used, thecombinations used in amplification, and the expected sizes of theamplification products are illustrated in FIG. 18 (within homologousregion). In the primer set designed within the homologous recombinationregion, 3046 bp was amplified in the Parietichytrium sarkarianum SEK364strain, and 2605 bp was amplified in the Δ4 desaturase gene disruptionstrain. [RHO251: 20mer: 5′-GTG GTC GAA GTG GAG TAT CT-3′ (SEQ ID NO:49). RHO252: 20mer: 5′-ACT CGC CAT ACA ACT TTA CA-3′ (SEQ ID NO: 50)].

As a result, a Δ4 desaturase gene disruption strain in which there is noamplification derived from the wild-type allele (Wt allele) and there isamplification derived from the Δ4 desaturase gene KO allele (NeoRallele) was obtained (FIG. 19 lane 7: Δ4 desaturase KO mutant strain).

Example 3-6: Change in Fatty Acid Composition by Δ4 Desaturase GeneDisruption

The Parietichytrium sarkarianum SEK364 wild-type strain and the Δ4desaturase gene disruption strain thereof were cultured according to themethod described in Example 2-9, and after freeze drying, the fattyacids were methyl-esterified and analyzed using GC. In culturing, the GYliquid culture medium described in Example 1 supplemented with 0.1% of avitamin solution (vitamin B₁ 200 mg, vitamin B₂ 1 mg, and vitamin B₁₂ 1mg are dissolved in 100 mL of distilled water) and 0.2% of a traceelement solution (EDTA disodium salt 30.0 g, FeCl₃.6H₂O 1.45 g, H₂BO₃34.2 g, MnCl₂.4H₂O 4.3 g, ZnCl₂ 1.335 g, CoCl₂.6H₂O 0.13 g, NiSO₄.6H₂O0.26 g, CuSO₄.5H₂O 0.01 g, and NaMoO₄.2H₂O 0.025 g are dissolved in 1 Lof distilled water) was used. In GC analysis, measurement was performedusing a gas chromatograph GC-2014 (available from Shimadzu Corporation)under the following conditions. Column: HR-SS-10 (30 m×0.25 mm;available from Shinwa Chemical Industries Ltd.); column temperature:150° C.→(2° C./min)→220° C. (10 min); carrier gas: He (1.3 mL/min).

The analysis results chart is shown in FIG. 20, and a partial enlargeddiagram thereof is shown in FIG. 21. FIG. 22 shows a quantification ofthe chart of FIG. 20. This table shows a comparison of the fatty acidcompositions of the Parietichytrium sarkarianum SEK364 wild-type strainand the Δ4 desaturase gene disruption strain thereof. This table is thequantification of the chart of FIG. 20. The table shows that, of thetotal fatty acid composition, ARA is 1.59%, DGLA is 0.98%, ETA is 0.05%,EPA is 0.79%, n-6 DPA is 0.00%, and DHA is 0.00%. The table shows that,by GC area, LA/EPA is 5.09, GLA/EPA is 0.48, DTA/EPA is 7.44, DTA/ARA is3.73, DTA/DGLA is 6.06, DGLA/LA is 0.24, ARA/LA is 0.39, EPA/LA is 0.20,DTA/LA is 1.46, DGLA/GLA is 2.57, ARA/GLA is 4.19, and n-6 DPA/DTA is0.00.

These results show that when the Δ4 desaturase gene is disrupted in theParietichytrium sarkarianum SEK364 strain, DHA and DPA n-6 cannot besubstantially biosynthesized, and conversely, DPA n-3 and DTA, which aresubstrates thereof, increase.

By selecting the labyrinthulid Parietichytrium sarkarianum SEK364 havingno PUFA-PKS pathway in this manner, a strain that accumulates PUFAsother than DHA and DPA n-6 can be produced without PUFA-PKS pathway genedisruption. This strain may also be used as a strain that produces n-3DPA and/or DTA, and further disruption or transforming elongase ordesaturase genes can create strains that produce desired PUFAs.

Example 4 Measurement of Fatty Acid Composition of Lipids Produced byC20 Elongase Gene Disruption and Transformation Strain ofParietichytrium Sp. SEK358 [Example 4-1]: Transfer of C20 Elongase GeneTargeting Vector to Parietichytrium sp. SEK358 Strain

Using the targeting vector produced with pRH85 (FIG. 9) described inExample 2-6 as a template, the gene was amplified with PrimeSTAR Max DNAPolymerase (available from Takara Bio Inc.) using RHO153 (described inExample 2-4, SEQ ID NO: 24) and RHO1154 (described in Example 2-4. SEQID NO: 25) as primers. [PCR cycles: 98° C. 2 min/98° C. 30 sec, 68° 2min, 30 cycles/68° C. 2 min]. After phenol chloroform extraction andchloroform extraction, the DNA underwent ethanol precipitation, and theprecipitate was dissolved in 0.1×TE. A260/280 was measured and the DNAconcentration was calculated. The transfer fragment obtained when pRH85(FIG. 9) described in Example 2-6 was used as a template was 2661 bp,and resulted in a sequence composed of genus Parietichytrium C20elongase gene front half—SV40 terminator sequence—artificiallysynthesized neomycin resistance gene sequence—ubiquitin promotersequence—genus Parietichytrium C20 elongase gene back half (described inExample 2-7, SEQ ID NO: 31).

The Parietichytrium sp. SEK358 strain was cultured for 3 days in a GYculture medium, and cells in the logarithmic growth phase were used forgene transfer. To cells corresponding to OD600=1 to 1.5, 0.625 μg of DNAfragment was transformed by the gene gun method (microcarrier: 0.6micron gold particles, target distance: 6 cm, chamber vacuum: 26 mmHg,rupture disk: 900 psi). After a recovery time of 24 hr, the transgeniccells were spread on a PDA agar plate culture medium containing 0.5mg/mL of G418. As a result, from 10 to 30 cells of drug resistant strainper shot were obtained.

[Example 4-2]: Identification of C20 Elongase Gene Targeting HomologousRecombinant

Genome DNA of the Parietichytrium sp. SEK358 strain and the C20 elongasegene disruption strain were extracted by the method described in Example2-2, and then A260/280 was measured and the DNA concentration wascalculated. Using the genome DNA as templates, PCR for genome structureconfirmation was performed using Mighty Amp DNA Polymerase (availablefrom Takara Bio Inc.). The positions of the primers used, thecombinations used in amplification, and the expected sizes of theamplification products are illustrated in FIG. 10 described in Example2-8.

RHO184 (described in Example 2-8, SEQ ID NO: 37) was set upstream of C20elongase; RHO185 (described in Example 2-8, SEQ ID NO: 38) was setdownstream; RHO142 (described in Example 2-8, SEQ ID NO: 35) and RHO143(described in Example 2-8, SEQ ID NO: 36) were set on the artificiallysynthesized neomycin resistance gene. [PCR cycles: 98° C. 2 min/98° C.10 sec, 68° 2 min, 30 cycles/68° C. 7 min].

A C20 elongase gene disruption strain in which there is no amplificationin the wild-type allele (Wt allele) and there is amplification in theartificially synthesized neomycin resistance gene allele (NeoR allele)was obtained (FIG. 23).

[Example 4-3]: Change in Fatty Acid Composition by C20 Elongase GeneDisruption

The Parietichytrium sp. SEK358 wild-type strain and the gene disruptionstrain thereof (C20 elongase gene disruption strain, C20 KO) werecultured according to the method described in Example 2-9, and afterfreeze drying, the fatty acids were methyl-esterified and analyzed usingGC. In GC analysis, measurement was performed using a gas chromatographGC-2014 (available from Shimadzu Corporation) under the followingconditions. Column: HR-SS-10 (30 m×0.25 mm; available from ShinwaChemical Industries Ltd.); column temperature: 150° C.→(5° C./min)→220°C. (10 min); carrier gas: He (1.3 mL/min). The changes in the fatty acidcomposition are shown in FIG. 24. Furthermore, FIG. 25 shows theproportion when the wild-type strain is taken as 100%.

FIG. 25 shows that, of the total fatty acid composition, ARA is 21.35%,DGLA is 8.64%, ETA is 2.14%, EPA is 23.83%, n-6 DPA is 0.46%, and DHA is0.94%. FIG. 25 shows that, by GC area, LA/DHA is 4.6, GLA/DHA is 2.8,DGLA/DHA is 9.19, ARA/DHA is 22.7, EPA/DHA is 25.4, LA/EPA is 0.18,GLA/EPA is 0.11, DTA/EPA is 0.01, DTA/ARA is 0.01, DTA/DGLA is 0.03,LA/n-6 DPA is 9.3. GLA/n-6 DPA is 5.7. DGLA/n-6 DPA is 18.8, R/n-6 DPAis 46.4, EPA/n-6 DPA is 51.8, DGLA/LA is 2.0, ARA/LA is 5.0, EPA/LA is5.6, DTA/LA is 0.06, DGLA/GLA is 3.3, ARA/GLA is 8.2, n-6 DPA/DTA is1.8, DHA/n-3 DPA is 4.1. C20 PUFA/C22 PUFA is 29.61, and n-6 PUFA/n-3PUFA is 1.1.

As a result, when the C20 elongase gene was disrupted in theParietichytrium sp. SEK358 strain, fatty acids having not less than 22carbon chains decreased while fatty acids having 20 carbon chainsincreased. Specifically, arachidonic acid increased approximately 7-foldand EPA increased approximately 11-fold, while DPA decreased toapproximately 1/15 and DHA decreased to approximately ⅛.

By selecting the labyrinthulid Parietichytrium sp. SEK358 having noPUFA-PKS pathway in this manner, a strain that accumulates PUFAs otherthan DHA and DPA n-6 can be produced without PUFA-PKS pathway genedisruption. This strain may also be used as a strain that produces EPAand/or ARA, and further disruption or transforming elongase ordesaturase genes can create strains that produce desired PUFAs.

Example 5 Measurement of Fatty Acid Composition of Lipids Produced by Δ4Desaturase Gene Disruption and Transformation Strain of Parietichytriumsp. SEK358 [Example 5-1]: Transfer of Δ4 Desaturase Gene TargetingVector to Parietichytrium sp. SEK358 Strain

Using the targeting vector produced with pRHI26 (FIG. 17) described inExample 3-3 as a template, the gene was amplified with PrimeSTAR Max DNAPolymerase (available from Takara Bio Inc.) using RHO241 (described inExample 3-1, SEQ ID NO: 40) and RHO242 (described in Example 3-1, SEQ IDNO: 41) as primers. After phenol chloroform extraction and chloroformextraction, the DNA underwent ethanol precipitation, and the precipitatewas dissolved in 0.1×TE. A260/280 was measured and the DNA concentrationwas calculated. The transfer fragment obtained when pRHI26 (FIG. 17)described in Example 3-3 was used as a template was 4562 bp.

The Parietichytrium sp. SEK358 strain was cultured for 1 to 2 days in aGY culture medium, and cells in the logarithmic growth phase were usedin gene transfer. To cells corresponding to OD600=1 to 2, 0.625 μg ofDNA fragment was transformed by the gene gun method (microcarrier: 0.6micron gold particles, target distance: 6 cm, chamber vacuum: 26 mmHg,rupture disk: 1550 psi). After a recovery time of 24 hr, the transgeniccells were spread on a PDA agar plate culture medium containing 1 mg/mLof G418. As a result, from 0 to 2 cells of drug resistant strain pershot were obtained.

[Example 5-2]: Identification of Δ4 Desaturase Gene Targeting HomologousRecombinant

Genome DNA from the Parietichytrium sp. SEK358 strain and the Δ4desaturase gene disruption strain were extracted by the method describedin Example 2-2, and then A260/280 was measured and the DNA concentrationwas calculated. Using the genome DNA as templates, PCR for genomestructure confirmation was performed using PrimeSTAR GXL DNA Polymerase(available from Takara Bio Inc.). The positions of the primers used, thecombinations used in amplification, and the expected sizes of theamplification products are illustrated in FIG. 18 (within homologousregion) and FIG. 26 (outside homologous region). In the primer setdesigned within the homologous recombination region, 3046 bp wasamplified in the Parietichytrium sp. SEK358 strain, and 2605 bp wasamplified in the Δ4 desaturase gene disruption strain. [RHO251: 20mer:5′-GTG GTC GAA GTG GAG TAT CT-3′ (SEQ ID NO: 49), RHO252: 20mer: 5′-ACTCGC CAT ACA ACT TTA CA-3′ (SEQ ID NO: 50)]. In the primer set designedoutside the homologous recombination region, 5231 bp was amplified inthe Parietichytrium sp. SEK358 strain, and 4790 bp was amplified in theΔ4 desaturase gene disruption strain. [HGO32: 25mer: 5′-CGG AGC TCG GAGAAC AAC ATA GAA G-3′ (SEQ ID NO: 51), HGO33: 23mer: 5′-GTG CAA CCA GGTGGC AAG ATT GT-3′ (SEQ ID NO: 52)].

As a result, a Δ4 desaturase gene disruption strain in which there is noamplification derived from the wild-type allele (Wt allele) and there isamplification derived from the Δ4 desaturase gene KO allele (NeoRallele) was obtained (FIG. 27 lane 4, FIG. 28 lane 4: Δ4 desaturase KOmutant strain).

[Example 5-3]: Change in Fatty Acid Composition by Δ4 Desaturase GeneDisruption

The Parietichytrium sp. SEK358 wild-type strain and the Δ4 desaturasegene disruption strain thereof (SEK358 delta4 des. KO mutant strain)were cultured according to the method described in Example 2-9, andafter freeze drying, the fatty acids were methyl-esterified and analyzedusing GC. In culturing, the GY liquid culture medium described inExample 1 supplemented with 0.1% of a vitamin solution (vitamin B₁ 200mg, vitamin B₂ 1 mg, and vitamin B₁₂ 1 mg are dissolved in 100 mL ofdistilled water) and 0.2% of a trace element solution (EDTA disodiumsalt 30.0 g, FeCl₃.6H₂O 1.45 g, H₂BO₃ 34.2 g. MnCl₂.4H₂O 4.3 g, ZnCl₂1.335 g, CoCl₂.6H₂O 0.13 g, NiSO₄.6H₂O 0.26 g. CuSO₄.5H₂O 0.01 g. andNaMoO₄.2H₂O 0.025 g are dissolved in 1 L of distilled water) was used.In GC analysis, measurement was performed using a gas chromatographGC-2014 (available from Shimadzu Corporation) under the followingconditions. Column: HR-SS-10 (30 m×0.25 mm; available from ShinwaChemical Industries Ltd.); column temperature: 150° C.→(2° C./min)→220°C. (10 min); carrier gas: He (1.3 mL/min).

The analysis results chart is shown in FIG. 29, and a partial enlargeddiagram of the chart is shown in FIG. 30. The table in FIG. 31 shows aquantification of the chart of FIG. 29. This table shows a comparison ofthe fatty acid compositions of the Parietichytrium sp. SEK358 wild-typestrain and the Δ4 desaturase gene disruption strain thereof. The tableshows that, of the total fatty acid composition, ARA is 3.03%, DGLA is1.35%. ETA is 0.03%, EPA is 1.10%, n-6 DPA is 0.00%, and DHA is 0.00%.FIG. 31 shows that, by GC area, LA/EPA is 4.2, GLA/EPA is 0.71, DTA/EPAis 7.19, DTA/ARA is 2.60, DTA/DGLA is 5.85, DGLA/LA is 0.29, ARA/LA is0.66, EPA/LA is 0.24, DTA/LA is 1.71, DGLA/GLA is 1.72, ARA/GLA is 3.87,C20 PUFA/C22 PUFA is 0.42, and n-6 PUFA/n-3 PUFA is 2.0.

The results showed that when the Δ4 desaturase gene was disrupted in theParietichytriumn sp. SEK358 strain. DHA and DPA n-6 cannot besubstantially biosynthesized, and conversely, n-3 DPA and DTA, which aresubstrates thereof, increase.

By selecting the labyrinthulid Parietichytrium sp. SEK358 having noPUFA-PKS pathway in this manner, a strain that accumulates PUFAs otherthan DHA and n-6 DPA can be produced without PUFA-PKS pathway genedisruption. This strain may also be used as a strain that produces n-3DPA and/or DTA, and further disruption or transforming elongase ordesaturase genes can create strains that produce desired PUFAs.

Example 6 Measurement of Fatty Acid Composition of Lipids Produced byC20 Elongase Gene Disruption and Transformation Strain ofParietichytrium sp. SEK571 [Example 6-1]: Transfer of C20 Elongase GeneTargeting Vector to Parietichytrium sp. SEK571 Strain

Using the targeting vector produced with pRH85 (FIG. 9) described inExample 2-6 as a template, the gene was amplified with PrimeSTAR Max DNAPolymerase (available from Takara Bio Inc.) using RHO153 (described inExample 2-4, SEQ ID NO: 24) and RHO154 (described in Example 2-4, SEQ IDNO: 25) as primers. [PCR cycles: 98° C. 2 min/98° C. 30 sec, 68° C. 2min, 30 cycles/68° C. 2 min]. After phenol chloroform extraction andchloroform extraction, the DNA underwent ethanol precipitation, and theprecipitate was dissolved in 0.1×TE. A260/280 was measured and the DNAconcentration was calculated. The transfer fragment obtained when pRH85(FIG. 9) described in Example 2-6 was used as a template was 2661 bp,and resulted in a sequence including genus Parietichytrium C20 elongasegene front half-SV40 terminator sequence—artificially synthesizedneomycin resistance gene sequence—ubiquitin promoter sequence—genusParietichytrium C20 elongase gene back half (described in Example 2-7,SEQ ID NO: 31).

The Parietichytrium sp. SEK571 strain was cultured for 3 days in a GYculture medium, and cells in the logarithmic growth phase were used ingene transfer. To cells corresponding to OD600=1 to 1.5, 0.625 μg of DNAfragment was transformed by the gene gun method (microcarrier: 0.6micron gold particles, target distance: 6 cm, chamber vacuum: 26 mmHg,rupture disk: 1550 psi). After a recovery time of 24 hr, the transgeniccells were spread on a PDA agar plate culture medium containing 0.5mg/mL of G418. As a result, from 5 to 15 cells of drug resistant strainper shot were obtained.

[Example 6-2]: Identification of C20 Elongase Gene Targeting HomologousRecombinant

Genome DNA of the Parietichytrium sp. SEK571 strain and the C20 elongasegene disruption strain were extracted by the method described in Example2-2, and then A260/280 was measured and the DNA concentration wascalculated. Using the genome DNA as templates, PCR for genome structureconfirmation was performed using Mighty Amp DNA Polymerase (availablefrom Takara Bio Inc.). The positions of the primers used, thecombinations used in amplification, and the expected sizes of theamplification products are illustrated in FIG. 10 described in Example2-8.

RHO184 (described in Example 2-8, SEQ ID NO: 37) was set upstream of C20elongase; RHO185 (described in Example 2-8. SEQ ID NO: 38) was setdownstream; RHO142 (described in Example 2-8, SEQ ID NO: 35) and RHO143(described in Example 2-8, SEQ ID NO: 36) were set on the artificiallysynthesized neomycin resistance gene. [PCR cycles: 98° C. 2 min/98° C.10 sec, 68° 2 min, 30 cycles/68° C. 7 min].

A C20 elongase gene disruption strain in which there is no amplificationin the wild-type allele (Wt allele) and there is amplification in theartificially synthesized neomycin resistance gene allele (NeoR allele)was obtained (FIG. 32).

[Example 6-3]: Change in Fatty Acid Composition by C20 Elongase GeneDisruption

The Parietichytrium sp. SEK571 strain and the gene disruption strainthereof (C20 elongase gene disruption strain, C20 KO) were culturedaccording to the method described in Example 2-9, and after freezedrying, the fatty acids were methyl-esterified and analyzed using GC. InGC analysis, measurement was performed using a gas chromatograph GC-2014(available from Shimadzu Corporation) under the following conditions.Column: HR-SS-10 (30 m×0.25 mm; available from Shinwa ChemicalIndustries Ltd.); column temperature: 150° C.→(5° C./min)→220° C. (10min); carrier gas: He (1.3 mL/min).

The changes in the fatty acid composition are shown in FIG. 33.Furthermore, FIG. 34 shows the proportion when the wild-type strain istaken as 100%.

FIG. 34 shows that, of the total fatty acid composition, ARA is 13.24%,DGLA is 1.93%, ETA is 1.14%, EPA is 29.58%, n-6 DPA is 0.96%, and DHA is1.17%. FIG. 34 shows that, by GC area, LA/DHA is 1.5, GLA/DHA is 0.9,DGLA/DHA is 1.65, ARA/DHA is 11.3, EPA/DHA is 25.3, LA/EPA is 0.06,GLA/EPA is 0.04, DTA/EPA is 0.01, DTA/ARA is 0.01, DTA/DGLA is 0.08,LA/n-6 DPA is 1.8, GLA/n-6 DPA is 1.1, DGLA/n-6 DPA is 2.0, ARA/n-6 DPAis 13.8, EPA/n-6 DPA is 30.8, DGLA/LA is 1.1, ARA/LA is 7.8. EPA/LA is17.4, DTA/LA is 0.09, DGLA/GLA is 1.8, ARA/GLA is 12.4, n-6 DPA/DTA is6.4, DHA/n-3 DPA is 4.7. C20 PUFA/C22 PUFA is 18.1, and n-6 PUFA/n-3PUFA is 0.51.

As a result, when the C20 elongase gene was disrupted in theParietichytrium sp. SEK571 strain, fatty acids having not less than 22carbon chains decreased while fatty acids having 20 carbon chainsincreased. Specifically, arachidonic acid increased approximately 4-foldand EPA increased approximately 8-fold, while DPA decreased toapproximately 1/12 and DHA decreased to approximately 1/12.

The results show that Parietichytrium sp. SEK571 has very weak or noPUFA production activity via the PUFA-PKS pathway, and by selecting thelabyrinthulid Parietichytrium sp. SEK571, a strain that accumulatesPUFAs other than DHA and n-6 DPA can be produced without PUFA-PKSpathway gene disruption. This strain may also be used as a strain thatproduces EPA and/or ARA, and further disruption or transforming elongaseor desaturase genes can create strains that produce desired PUFAs.

Comparative Example 1 Measurement of Fatty Acid Composition of LipidsProduced by C20 Elongase Gene Disruption and Transformation Strain ofThraustochytrium aureum ATCC 34304 [Comparative Example 1-1]: Extractionof Total RNA Derived from T. Aureum ATCC 34304, and mRNA Purification

A T. aureum ATCC 34304 culture solution on the third day of culturingusing a GY liquid culture medium was centrifuged for 15 min at 3500×g,and the cells were collected. The obtained cells were washed bysuspending in a sterilized physiological saline solution and thencentrifuging again, and were then rapidly frozen with liquid nitrogen,and then ground into powder form in a mortar. Total RNA was extractedfrom the obtained crushed cell solution using Sepasol-RNA I Super(available from Nacalai Tesque, Inc.). Then, mRNA was purified from thetotal RNA according to manufacturer's instructions using Oligotex-dT30<Super> mRNA Purification Kit (trade name; available from Takara BioInc.). The obtained total RNA and mRNA were dissolved in an appropriateamount of TE, and then subjected to electrophoresis usingformalin-modified gel (1% agarose/MOPS buffer). The result showed thattotal RNA extraction was successful, that mRNA was purified from thetotal RNA, and that the RNA was not decomposed by RNase. Furthermore, toproactively avoid RNA decomposition, rubber gloves, a mask, and the likewere donned through the experimental operation, and the instruments usedwere completely RNase-free or the RNase used was inactivated bytreatment with diethylpyrocarbonate (available from Nacalai Tesque,Inc.). Furthermore, when decomposing RNA, a solution obtained by addingthe recombinant RNase inhibitor RNaseOUT (trade name; available fromInvitrogen Corp.) to sterilized MilliQ water treated withdiethylpyrocarbonate was used.

[Comparative Example 1-2]: Isolation of T. Aureum ATCC 34304-DerivedElongase Gene by RACE

Using a histidine box (His box) in which the elongase gene was conservedto a high degree as a target, forward (elo-F: 5′-TTY YTN CAY GTN TAY CAYCAY-3′) (SEQ ID NO: 53) and reverse (elo-R, 5′-GCR TGR TGR TAN ACR TGNARR AA-3′) (SEQ ID NO: 54) degenerate oligonucleotides were synthesized.The oligonucleotides were synthesized using a DNA synthesizer (availablefrom Applied Biosystems Corp.). Next, 3′- and 5′-RACE cDNA libraries inwhich synthetic adapters were appended to the 3′ and 5′ terminals wereproduced according to manufacturer's instructions using SMART RACE cDNAAmplification Kit (trade name; available from Clontech Laboratories,Inc.). Using these as templates, 3′- and 5′-RACE were performed usingthe synthetic adapter-specific oligonucleotides and the above degenerateoligonucleotides elo-F and elo-R. [PCR cycles: 94° C. 1 min/94° C. 30sec, 60° C. 30 sec, 72° C. 3 min, 30 cycles/72° C. 10 min/4° C. ∞]. As aresult, bands of specifically amplified 3′- and 5′-RACE products wereconfirmed (FIG. 35). Next, the entire amounts of the RACE products weresubjected to electrophoresis using 1% agarose gel, the separated DNAfragments were cut with a clean cutter or the like, and DNA fragmentswere extracted according to the method described in Non-patent Document10. Then, TA cloning of the DNA fragments was performed using pGEM-TEasy Vector (available from Promega Corporation), and the base sequencesthereof were determined according to the method of Sanger et al.(Non-patent Document 11). Specifically, using BigDye (trade name)Terminator v3.1 Cycle Sequencing Kit and 3130 Genetic Analyzer(available from Applied Biosystems Corp.), the base sequence wasdetermined by the dye terminator method according to manufacturer'sinstructions.

As a result, two respective sequences of 190 bp and 210 bp named elo1(SEQ ID NO: 55) and elo2 (SEQ ID NO: 56) were successfully identified inthe 3′-RACE product, and one 200 bp sequence named elo3 (SEQ ID NO: 57)was successfully identified in the 5′-RACE product. The fact that theseelo1, elo2, and elo3 sequences exhibited significant homology to variouselongase gene sequences shows that these sequences are partial sequencesof elongase genes derived from T. aureum ATCC 34304. Additionally,respective oligonucleotide primers were again designed for elo1, elo2,and elo3, and acquisition of cDNA sequences was attempted by RACE. Theproduced oligonucleotide primers are shown below, elo1 forwardoligonucleotide primer (elo1-F1; 5′-TAT GAT CGC CAA GTA CGC CCC-3′) (SEQID NO: 58) and reverse oligonucleotide primer (elo1-R1; 5′-GAA CTG CGTCAT CTG CAG CGA-3′) (SEQ ID NO: 59), elo2 forward oligonucleotide primer(elo2-F1; 5′-TCT CGC CCT CGA CCA CCA AC-3′) (SEQ ID NO: 60) and reverseoligonucleotide primer (elo2-R1: 5′-CGG TGA CCG AGT TGA GGT AGC C-3′)(SEQ ID NO: 61), elo3 forward oligonucleotide primer (elo3-F1; 5′-CAACCC TTT CGG CCT CAA CAA G-3′) (SEQ ID NO: 62) and reverseoligonucleotide primer (elo3-R1; 5′-TTC TTG AGG ATC ATC ATG AAC GTGTC-3′) (SEQ ID NO: 63).

Using the produced forward and reverse oligonucleotide primers, RACE andbase sequence analysis of the amplified products were performed by thesame methods as described above. As a result, for elo1, specificallyamplified 3′- and 5′-RACE products were obtained, and since theduplicate portions thereof matched completely, they were proved to be an1139 bp elo1 cDNA sequence (SEQ ID NO: 64). Similarly, for elo3,specifically amplified 3′- and 5′-RACE products were obtained, and sincethe duplicate portions thereof matched completely, they were proved tobe a 1261 bp elo3 cDNA sequence (SEQ ID NO: 65).

As a result of sequence analysis, it was found that elo1 is composed ofan 825 bp translation sequence (SEQ ID NO: 67) that encodes 275 aminoacid residues (SEQ ID NO: 66), and as a result of a BLAST search, it wasfound not only that elo1 exhibited significant homology to variouselongase genes, but also that elo1 completely matches a known presumedΔ5 elongase gene sequence derived from T. aureum (NCBI accession no.CS486301). On the other hand, elo3 was estimated to constituted of a 951bp translation region (SEQ ID NO: 69) that encodes 317 amino acidresidues (SEQ ID NO: 68), and as a result of a BLAST search, elo3 wasfound to exhibit significant homology to various elongase genes, andthus was confirmed to be a presumed elongase gene derived from T. aureumATCC 34304. Furthermore, a His box in which the elongase gene wasconserved to a high degree was found within the presumed amino acidsequences of the two genes. From the above results, the elo1 and elo3gene were considered to be presumed elongase genes derived from T.aureum ATCC 34304 and were designated as TaELO1 and TaELO2,respectively.

[Comparative Example 1-3]: Expression of TaELO1 and TaELO2 with Brewer'sYeast Saccharomyces cerevisiae as Host, and Analysis of Fatty AcidComposition of Transgenic Strains

Respective expression vectors were constructed in order to causeexpression of TaELO1 and TaELO2 using brewer's yeast S. cerevisiae as ahost. An outline thereof is given below. A pair of oligonucleotideprimers (E1 HindIII; 5′-ATA AGC TTA AAA TGT CTA GCA ACA TGA GCG CGT GGGGC-3′) (SEQ ID NO: 70) and (E1 XbaI; 5′-TGT CTA GAA CGC GCG GAC GGT CGCGAA A-3′) (SEQ ID NO: 71) were produced based on the sequence of theTaELO1 translation region. E1 HindIII is a forward oligonucleotideprimer and has a restriction enzyme HindIII site (AAGCTT) at the 5′terminal. Furthermore, the sequence near the start codon of TaELO1 hasbeen modified in reference to the yeast consensus sequence ((A/Y) A(A/U) A AUG UCU: underlined portion is start codon) (Non-patent Document12). E1 XbaI is a reverse oligonucleotide primer, and has an XbaI site(TCTAGA) at the 5′ terminal.

Similarly, a pair of oligonucleotide primers (E2 HindIII; 5′-TAA AGC TTAAAA TGT CTA CGC GCA CCT CGA AGA GCG CTC C-3′) (SEQ ID NO: 72) and (E2XbaI; 5′-CAT CTA GAC TCG GAC TTG GTG GGG GCG CTT G-3′) (SEQ ID NO: 73)were produced based on the sequence of the TaELO2 translation region. E2HindIII is a forward oligonucleotide primer and has a restriction enzymeHindIII site at the 5′ terminal. Furthermore, the sequence near thestart codon of TaELO2 has been modified in reference to the yeastconsensus sequence. E2 XbaI is a reverse oligonucleotide primer, and hasan XbaI site at the 5′ terminal.

With the 5′-RACE cDNA library described in Comparative Example 1-2 as atemplate, PCR was performed using the above two oligonucleotide primerpairs. A 949 bp TaELO1 translation region (SEQ ID NO: 74) and a 967 bpTaELO2 translation region (SEQ ID NO: 75), in which the vicinity of thestart codon was modified to the consensus sequence and having therestriction enzyme HindIII at the 5′ terminal and the restriction enzymeXbaI site at the 3′ terminal, were amplified. Furthermore, to avoidelongation mistakes, PrimeSTAR DNA Polymerase (trade name; availablefrom Takara Bio Inc.) having high correction activity was used. [PCRcycles: 98° C. 2 min/98° C. 5 sec, 60° C. 5 sec, 72° C. 1.5 min, 30cycles/72° C. 7 min/4° C. ∞].

Next, the amplified PCR products were separated with 1% agarose gel, andthen DNA fragments were cut and extracted from the agarose gel.Additionally, after treatment with restriction enzymes Hindi and XbaI,the DNG fragments were again purified using agarose gel, and a cyclizedvector was constructed by ligation using DNA Ligation Kit “Mighty Mix”(available from Takara Bio Inc.) to a brewer's yeast expression vectorpYES2/CT (available from Invitrogen Corp.) which was made into astraight chain by treatment with restriction enzymes HindIII and XbaI.Furthermore, by analyzing the base sequence, it was confirmed thatmutations due to PCR elongation mistakes had not been introduced intothe sequences of the TaELO1 and TaELO2 translation regions transformedinto pYES2/CT. From the above result, a TaELO1 expression vector pYEELO1and a TaELO2 expression vector pYEELO2 were successfully constructed.

Transformants in which the two constructed expression vectors andpYES2/CT had been transformed into brewer's yeast S. cerevisiae by thelithium acetate method were selected according to the methods describedin Non-patent Document 13 and Non-patent Document 14. Next, the obtainedtransformants (pYEELO1 transgenic strain, pYEELO2 transgenic strain, andmock transgenic strain) were cultured according to the method of Qiu etal. (Non-patent Document 15), and cell-derived fatty acid extraction andmethyl-esterification were performed. However, culturing was carried outafter adding 0.02 mM each of the following acids in respective finalconcentrations: α-linolenic acid (ALA, C18:3Δ9, 12, 15) and linoleicacid (LA, C18:2Δ9, 12) as Δ9 elongase substrates; stearidonic acid (STA,C18:4Δ6, 9, 12, 15) and γ-linolenic acid (GLA, C18:3Δ6, 9, 12) as Δ6elongase substrates; eicosapentaenoic acid (EPA. C20:5Δ5, 8, 11, 14, 17)and arachidonic acid (AA, C20:4Δ5, 8, 11, 14) as Δ5 elongase substrates.Then, gas chromatography (GC) analysis of the methyl-esterified fattyacids was performed according to the method of Abe et al. (Non-patentDocument 16). In GC analysis, measurement was performed using a gaschromatograph GC-2014 (available from Shimadzu Corporation) under thefollowing conditions. Column: HR-SS-10 (30 m×0.25 mm; available fromShinwa Chemical Industries Ltd.); column temperature: 150° C.→(5°C./min)→220° C. (10 min), carrier gas: He (1.3 mL/min).

As a result, the pYEELO1 transgenic strain exhibited Δ6 elongaseactivity, by which stearidonic acid (STA) is converted toeicosatetraenoic acid (ETA, 20:4Δ8, 11, 14, 17) and γ-linolenic acid(GLA) is converted to dihomo-γ-linolenic acid (DGLA, C20:3Δ8, 11, 14),which was not seen in the host (mock transgenic strain). On the otherhand, the pYEELO1 transgenic strain exhibited Δ9 elongase activity, bywhich α-linolenic acid (ALA) is converted to eicosatrienoic acid (ETrA.C20:3Δ11, 14, 17) and linoleic acid (LA) is converted to eicosadienoicacid (EDA, C20:3Δ1, 14), as well as Δ5 elongase (=C20 elongase)activity, by which eicosapentaenoic acid (EPA) is converted to ω3docosapentaenoic acid (ω3 DPA, C22:5Δ7, 10, 13, 16, 19) and arachidonicacid (ARA) is converted to docosatetraenoic acid (DTA, C22: 4Δ7, 10, 13,16) (Table 1).

Furthermore, the pYEELO2 transgenic strain exhibited Δ5 elongase (=C20elongase) activity by which EPA is converted to 03 DPA (C22: 5Δ7, 10,13, 16, 19) and ARA is converted to DTA, which was not seen in the host.On the other hand, the pYEELO2 transgenic strain exhibited slight Δ6elongase activity, by which STA is converted to ETA and GLA is convertedto DGLA (Table 1). The above result shows that TaELO1 is Δ6/Δ9/Δ5elongase, and TaELO2 is Δ5/Δ6 elongase.

TABLE 1 LA supplemented (0.2 mM) ALA supplemented (0.2 mM) mock TaELO1TaELO2 mock TaELO1 TaELO2 LA 30.5 23.5 36.3 ALA 49.1 25.8 47.1 EDA 0.28.9 0.2 ETrA 0.2 17.9 0.3 Conversion 27.4 Conversion 41 efficiencyefficiency (%) (%) GLA supplemented (0.2 mM) STA supplemented (0.2 mM)mock TaELO1 TaELO2 mock TaELO1 TaELO2 GLA 44.0 7.6 43.6 STA 46.2 8.340.5 DGLA 0.2 29.0 0.8 ETA 0.3 28.1 1.7 Conversion 79.3 1.9 Conversion77.2 4.0 efficiency efficiency (%) (%) ARA supplemented (0.2 mM) EPAsupplemented (0.2 mM) mock TaELO1 TaELO2 mock TaELO1 TaELO2 ARA 30.923.2 8.9 EPA 42.0 31.2 13.1 ADA — 5.8 13.6 DPA 0.1 10.6 24.5 Conversion20.1 60.3 Conversion 25.3 65.1 efficiency efficiency (%) (%) Conversionefficiency (%) = 100 × product (area)/substrate (area) + product (area)(n = 1)

[Comparative Example 1-4]: Acquisition of TaELO2 ORF Upstream andDownstream Regions by PCR Genome Walking

In the targeting vector for TaELO2 disruption, the regions upstream anddownstream of TaELO2 ORF serving as homologous recombination sites wereacquired by PCR genome walking. An overview is given below.

Cells of the T. aureum ATCC 34304 strain on the third day of culturingin a GY liquid culture medium were rapidly frozen with liquid nitrogen,and then ground into powder form in a mortar. After genome DNA wasextracted according to the method described in Non-patent Document 17,and was dissolved in an appropriate quantity of TE. The quantity andpurity of the genome DNA were tested by O.D. 260 and O.D. 280measurement. Next, a genome DNA library was constructed, wherein acassette sequence having restriction enzyme sites was appended to genomeDNA cut with various restriction enzymes according to the manufacturer'sprotocol using TaKaRa LA PCR (trade name) in vitro Cloning Kit(available from Takara Bio Inc.). Then, using the produced genome DNAlibrary as a template, nested PCR was performed according to themanufacturer's protocol using forward oligonucleotide primers E2 XbaI(described in Comparative Example 1-3, SEQ ID NO: 73) produced based onthe sequence of TaELO2 and elo3-F1 (described in Comparative Example1-2, SEQ ID NO: 62), or the reverse oligonucleotide primers E2 HindIII(described in Comparative Example 1-3, SEQ ID NO: 72) and elo3-R1(described in Comparative Example 1-2, SEQ ID NO: 63), together witholigonucleotide primers complementary to the sequences of cassettesincluded in the kit. As a result, an 1122 bp TaELO2 ORF upstreamsequence (SEQ ID NO: 76) and a 1204 bp TaELO2 ORF downstream sequence(SEQ ID NO: 77) were successfully acquired.

[Comparative Example 1-5]: Construction of TaELO2 Targeting Vector withNeor as Selection Marker

A DNA fragment in which TaELO2 ORF upstream sequence/artificiallysynthesized Neor/TaELO2 ORF downstream sequence were ligated andproduced by fusion PCR. The oligonucleotide primers used were as shownbelow.

KO Pro F SmaI (SEQ ID NO: 78)(31mer: 5′-CTC CCG GGT GGA CCT AGC GCG TGT GTC ACC T-3′) Pro R(SEQ ID NO: 79) (25mer: 5′-GGT CGC GTT TAC AAA GCA GCG CAG C-3′) SNeo F(SEQ ID NO: 80) (52mer: 5′-GCT GCG CTG CTT TGT AAA CGC GAC CAT GATTGA ACA GGA CGG CCT TCA CGC T-3′) SNeo R (SEQ ID NO: 81)(52mer: 5′-TCG GGA GCC AGC CGG AAA CAG GTT CAA AAGAAC TCG TCC AGG AGG CGG TAG A-3′) Term F (SEQ ID NO: 82)(23mer: 5′-ACC TGT TTC CGG CTG GCT CCC GA-3′) KO Term R SmaI(SEQ ID NO: 83) (27mer: 5′-ATC CCG GGG CCG AGA ACG GGG TCG CCC-3′)

Of these oligonucleotide primers, KO Pro F SmaI/Pro R were used inamplification of the TaELO2 ORF upstream sequence using the T. aureumATCC 34304 genome DNA described in Comparative Example 1-4 as atemplate, SNeo F/SNeo R were used in amplification of artificiallysynthesized Neor using artificially synthesized Neor as a template, andTerm F/KO Term R SmaI were used in amplification of the TaELO2 ORFdownstream sequence using the T. aureum ATCC 34304 genome DNA describedin Comparative Example 1-4 as a template. As PCR conditions,denaturation was performed at 98° C. for 10 sec. and annealing and theelongation reaction were performed while adjusting as appropriateaccording to Tm of the primers and the lengths of the amplificationproducts.

As a result, a 2696 bp (SEQ ID NO: 84) TaELO2 ORF upstreamsequence/artificially synthesized Neor/TaELO2 ORF downstream sequencewas successfully ligated. The result of TA cloning of this sequenceusing pGEM-T Easy Vector (available from Promega Corporation) was usedas a disruption vector, and was named pTKONeor.

[Comparative Example 1-6]: Transfer of TKONeor into T. Aureum ATCC 34304

Using, as a template, pTKONeor, which is the TaELO2 targeting vectorwith the artificially synthesized Neor as a selection marker produced inComparative Example 1-5, TaELO2 ORF upstream sequence/artificiallysynthesized Neor/TaELO2 ORF downstream sequence was amplified using apair of oligonucleotide primers KO Pro F SmaI (Comparative Example 1-5,SEQ ID NO: 78)/KO Term R SmaI (Comparative Example 1-5, SEQ ID NO: 83)and using PrimeSTAR (trade name) HS DNA Polymerase (available fromTakara Bio Inc.). [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 3 min,30 cycles/68° C. 10 min/4° C. ca]. After electrophoresis using 1%agarose gel, the DNA fragments were extracted, and after ethanolprecipitation, the extracted DNA were dissolved in an appropriatequantity of TE. The quantity and purity of the DNA fragments were testedby O.D. 260 and O.D. 280 measurement. The obtained DNA fragment iscalled TKONeor hereinafter.

Next, the introduction of DNA was performed by the gene gun method.Specifically, the T. aureum ATCC 34304 strain was cultured at 25° C. at150 rpm using a GY liquid culture medium, and cells of the middle tolatter logarithmic growth phase were centrifuged for 10 min at 4° C. at3500-g, and the supernatant was removed. Then, the obtained cells wereresuspended in a GY liquid culture medium so as to result in 100 timesthe concentration of the original culture solution. 20 μL of this cellsuspension was thinly spread uniformly and dried in a diameter ofapproximately 3 cm on PDA agar plate culture medium 5 cm in diametercontaining 1 mg/mL of G418 (available from Nacalai Tesque, Inc.). Usinga PDS-1000/He System (available from Bio-Rad Laboratories, Inc.),implantation was performed on under the conditions of target distance: 6cm, vacuum: 26 inches Hg, microcarrier size: 0.6 μm, rupture disk(implantation pressure): 1100 psi. After that, 100 μL of PD liquidculture medium was added drop-wise to the PDA agar plate culture medium,and the cells were spread out again and static culturing was performed.As a result, transformants conferred with G418 resistance were obtainedwith efficiency of 4.7×10¹ cfu/μg DNA.

[Comparative Example 1-7]: PCR Using Genome DNA of Transformant in whichTKONeor was Transformed as a Template

Seven colonies of transformants were extracted with a toothpick, andwere then inoculated in a GY liquid culture medium containing 0.5 mg/mLof G418 (available from Nacalai Tesque, Inc.). After subculturingmultiple times, genome DNA was extracted from the cells by the methoddescribed in Comparative Example 1-4, and after ethanol precipitation,the extracted genome DNA was dissolved in an appropriate amount of TE.The quantity and purity of the extracted genome DNA were tested by O.D.260 and O.D. 280 measurement. Then, with the obtained genome DNA of theobtained transformant and the wild-type as templates, PCR was performedusing various oligonucleotide primer pairs. The used oligonucleotideprimer pairs were as follows:

(1) Neor detection—Sneo F (described in Comparative Example 1-5, SEQ IDNO: 80) and SNeo R (described in Comparative Example 1-5, SEQ ID NO:81);

(2) KO confirmation 1—KO Pro F Sinai (described in Comparative Example1-5, SEQ ID NO: 78) and KO Term R SmaI (described in Comparative Example1-5, SEQ ID NO: 83);

(3) KO confirmation 2—E2 KO ProF EcoRV (30mer: 5′-GGA TAT CCC CCG CGAGGC GAT GGC TGC TCC-3′) (SEQ ID NO: 85) and SNeo R (described inComparative Example 1-5, SEQ ID NO: 81);

(4) KO confirmation 3—Sneo F (described in Comparative Example 1-5, SEQID NO: 80) and E2 KO Term R EcoRV (30mer: 5′-TGA TAT CGG GCC GCG CCC TGGGCC GTA GAT-3′) (SEQ ID NO: 86);

(5) TaELO2 amplification—E2 HindIII (described in Comparative Example1-3, SEQ ID NO: 72) and E2 XbaI (described in Comparative Example 1-3,SEQ ID NO: 73) (FIG. 36A).

As a result, it was confirmed that six of the seven analyzed clones weretransformants by random integration, but in one clone, TaELO2 ORF wassubstituted for Neor by homologous recombination (FIG. 36B, lanes 9,13). At the same time, however, it was found that TaELO2 ORF wasamplified (FIG. 36B, lane 17). This suggests the possibility that T.aureum ATCC 34304 is at least diploid or TaELO2 is a multicopy gene.

[Comparative Example 1-8]: Confirmation of TaELO2 Copy Number bySouthern Blotting

The following experiment was conducted in accordance with the methoddescribed in “DIG Manual [Japanese Edition] 8th, Roche Applied Science”(Non-patent Document 18). Specifically, wild-type genome DNA was cutwith various restriction enzymes and then subjected to electrophoresisusing 2.5 μg of 0.7% SeaKem (trade name) GTG (trade name) agarose(available from Takara Bio Inc.) per lane. This was transformed to anylon membrane (Hybond (trade name)-N+, available from GE HealthcareInc.), and hybridized at 48° C. for 16 hr with a DIG labeled probeproduced using PCR DIG Probe Synthesis Kit (available from Roche AppliedScience, Inc.). The pair of oligonucleotide primers used in producingthe DIG labeled probe were TaELO2 det F (25mer: 5′-GTA CGT GCT CGG TGTGAT GCT GCT C-3′) (SEQ ID NO: 87) and TaELO2 det R (24mer: 5′-GCG GCGTCC GAA CAG GTA GAG CAT-3′) (SEQ ID NO: 88). [PCR cycles: 98° C. 2min/98° C. 30 sec, 65° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 7min/4° C. c]. The hybridized probes were detected using the colordevelopment method (NBT/BCIP solution).

The results demonstrated that TaELO2 is a single copy gene from the factthat single bands were detected in all lanes treated with the variousrestriction enzymes (FIG. 37). This suggests that T. aureum ATCC 34304is at least diploid.

[Comparative Example 1-9]: Evaluation by Southern Blotting ofTransformant in which TKONeor was Transformed

Southern blotting was performed by the method described in ComparativeExample 1-8. Specifically, southern blotting was performed using thecolor development method (NBT/BCIP solution) relative to genome DNA ofthe transformant and a wild-type strain digested with EcoRV and PstI,using a DIG labeled probe amplified using the pair of oligonucleotideprimers uprobe F (35mer: 5′-ATC CGC GTA TAT ATC CGT AAA CAA CGG AAC ATTCT-3′) (SEQ ID NO: 89) and uprobe R (26mer: 5′-CTT CGG GTG GAT CAG CGAGCG ACA GC-3′) (SEQ ID NO: 90). [PCR cycles: 98° C. 2 min/98° C. 30 sec,65° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 7 min/4° C. ∞]. In thiscase, in the wild-type allele, a DNA fragment of approximately 1.2 kbpwas detected, but in the mutant allele in which TaELO2 ORF wassubstituted for Neor by homologous recombination, a DNA fragment ofapproximately 2.5 kbp was detected (FIG. 38(A)).

The result of analysis shows that T. aureum ATCC 34304 is at leastdiploid, since the wild-type allele band was also detected at the sametime as the mutant allele (FIG. 38(B)).

[Comparative Example 1-10]: Construction of TaELO2 Targeting Vector withHygr as Selection Marker

To disruption the remaining wild-type allele, a TaELO2 targeting vectorwith Hygr as a selection marker was constructed.

First, an ubiquitin promoter sequence derived from T. aureum ATCC 34304and Hygr were ligated by fusion PCR. The oligonucleotide primers usedwere as shown below.

ubi-600p F (SEQ ID NO: 91)(27mer: 5′-GCC GCA GCG CCT GGT GCA CCC GCC GGG-3′) ubi-hygro R(SEQ ID NO: 92) (59mer: 5′-TCG CGGG TGA GTT CAG GCT TTT TCA TGTTGG CTA GTG TTG CTT AGG TCG CTT GCT GCT G-3′) ubi-hygro F(SEQ ID NO: 93) (57mer: 5′-AGC GAC CTA AGC AAC ACT AGGC CAA CATGAA AAA GCC TGA ACT CAC CGC GAC GTC TG-3′) hygro R (SEQ ID NO: 94)(29mer: 5′-CTA TTC CTT TGC CCT CGG ACG AGT GCT GG-3′)

Of these oligonucleotide primers, ubi-600p F/ubi-hygro R were used inamplification of the T. aureum ATCC 34304-derived ubiquitin promotersequence using the T. aureum ATCC 34304 genome DNA described inComparative Example 1-4 as a template. Ubi-hygro F/hygro R were used inamplification of artificially synthesized Hygr using pcDNA 3.1 Zeo(available from Invitrogen Corp.) as a template. As PCR conditions,denaturation was performed at 98° C. for 10 sec, and annealing and theelongation reaction were performed while adjusting as appropriateaccording to Tm of the primers and the lengths of the amplificationproducts.

As a result, 1636 bp (SEQ ID NO: 95) of T. aureum ATCC 34304-derivedubiquitin promoter sequence/Hygr was successfully ligated. The result ofTA cloning of this sequence using pGEM-T Easy Vector (available fromPromega Corporation) was named pTub600Hygr.

Then, using pTub600Hygr as a template, PCR was performed using a pair ofoligonucleotide primers ubi-600p F NheI (33mer: 5′-GTG CTA GCC GCA GCGCCT GGT GCA CCC GCC GGG-3′) (SEQ ID NO: 96) and hygro R XbaI (37mer:5′-GTT CTA GAC TAT TCC TTT GCC CTC GGA CGA GTG CTG G-3′) (SEQ ID NO: 97)and using PrimeSTAR HS DNA Polymerase (available from Takara Bio Inc.).[PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 3 min, 30 cycles/68° C.10 min/4° C. ∞]. As a result, a T. aureum ATCC 34304-derived ubiquitinpromoter sequence/Hygr DNA fragment with NheI appended at the 5′terminal and an XbaI site appended at the 3′ terminal was prepared.Furthermore, using pTKONeor described in Comparative Example 1-5 as atemplate, PCR was performed using a pair of oligonucleotide primers KOvec F XbaI (37mer: 5′-GTT CTA GAC CTG TTT CCG GCT GGC TCC CGA GCC ATGC-3′) (SEQ ID NO: 98) and KO vec R NheI (40mer: 5′-GTG CTA GCG GTC GCGTTT ACA AAG CAG CGC AGC AAC AGA A-3′) (SEQ ID NO: 99) and usingPrimeSTAR HS DNA Polymerase (available from Takara Bio Inc.). [PCRcycles: 98° C. 2 min/98° C. 10 sec, 68° C. 3 min, 30 cycles/68° C. 10min/4° C. ∞]. As a result, a linear vector in which Neor of pTKONeordescribed in Comparative Example 1-5 was removed and having NheIappended at the 3′ terminal and an XbaI site appended at the 5′ terminalwas prepared. After the two DNA fragments were digested with NheI andXbaI, the DNA fragments were purified using agarose gel, and cyclicvectors were constructed using Ligation Convenience Kit (available fromNippon Gene Co., Ltd.).

The constructed TaELO2 targeting vector having Hygr as a selectionmarker includes pGEM-T Easy Vector (available from Promega Corporation)as a basic framework, and as an insertion sequence, had a 3537 bp (SEQID NO: 100) TaELO2 ORF upstream sequence/T. aureum ATCC 34304-derivedubiquitin promoter sequence/Hygr/TaELO2 ORF downstream sequence. Thiswas named pTKOub600Hygr.

[Comparative Example 1-11]: Evaluation of Transformant by PCR UsingKOub600Hygr Retransfer and Genome DNA as Templates, Southern Blotting,and RT-PCR

Using, as a template, pTKOub600Hygr (described in Comparative Example1-10), which is the constructed TaELO2 targeting vector with Hygr as aselection marker, TaELO2 ORF upstream sequence/T. aureum ATCC34304-derived ubiquitin promoter sequence/Hygr/TaELO2 ORF downstreamsequence was amplified using a pair of oligonucleotide primers KO Pro FSinai (Comparative Example 1-5, SEQ ID NO: 78)/KO Term R SmaI(Comparative Example 1-5, SEQ ID NO: 83) and using PrimeSTAR HS DNAPolymerase (available from Takara Bio Inc.). [PCR cycles: 98° C. 2min/98° C. 10 sec, 68° C. 3.5 min, 30 cycles/68° C. 10 min/4° C. ∞]. Theobtained DNA fragment was named KOub600Hygr. This was transformed intothe transformant obtained in Comparative Example 1-7 by the sametechnique, and after static culturing for 24 hr on PDA agar plateculture medium containing 1 mg/mL of G418 (available from NacalaiTesque. Inc.), the cells were collected. The static culturing wascontinued on PDA agar plate culture medium containing 1 mg/mL of G418(available from Nacalai Tesque, Inc.) and 2 mg/mL of hygromycin B(available from Wako Pure Chemical Industries, Ltd.) to obtain numeroustransformants (transfer efficiency: 1.02×10³ cfu/μg DNA).

Among them, 50 clones were extracted, and after subculturing multipletimes in a GY liquid culture medium containing 1 mg/mL of G418(available from Nacalai Tesque, Inc.) and 2 mg/mL of hygromycin B(available from Wako Pure Chemical Industries, Ltd.), genome DNA wasextracted by the same technique as described in Comparative Example 1-4,and after ethanol precipitation, the extracted genome DNA was dissolvedin an appropriate quantity of TE. The quantity and purity of theextracted genome DNA were tested by OD260 and OD280 measurement. Then,with the obtained transformant and wild-type genome DNA as templates,PCR was performed using various oligonucleotide primer pairs. [PCRcycles: 98° C. 2 min/98° C. 10 sec, 68° C. 1 min, 30 cycles/68° C. 10min/4° C. ∞]. The used oligonucleotide primer pairs were as follows:

(1) TaELO2 ORF detection—Sneo F (described in Comparative Example 1-5,SEQ ID NO: 80) and SNeo R (described in Comparative Example 1-5, SEQ IDNO: 81);

(2) KO confirmation—E2 KO Pro F EcoRV (described in Comparative Example1-7. SEQ ID NO: 85) and ubi-hygro R (described in Comparative Example1-10, SEQ ID NO: 92) (FIG. 39(A)).

The results showed that of the 50 analyzed clones, 14 clones weretransformants which caused homologous recombination in the form ofsubstituting TaELO2 ORF (FIG. 39(B), arrows). For these clones, it wasconfirmed that TaELO2 ORF was not amplified (FIG. 39(C)).

Then, southern blotting was performed using the technique described inComparative Example 1-9. Specifically, southern blotting was performedby the color development method (NBT/BCIP solution) relative to genomeDNA of the transformant and a wild-type strain digested with EcoRV andPstI, using a DIG labeled probe prepared using the pair ofoligonucleotide primers uprobe F (SEQ ID NO: 89) and uprobe R (SEQ IDNO: 90). In this case, in the wild-type allele, a DNA fragment ofapproximately 1.2 kbp was detected; in the mutant allele in which TaELO2ORF was substituted for Neor by homologous recombination, a DNA fragmentof approximately 2.5 kbp was detected; in the mutant allele in whichTaELO2 ORF was substituted for Hygr, a DNA fragment of approximately 1.9kbp was detected (FIG. 40(A)).

As a result of analysis, a band of the wild-type allele of approximately2.5 kbp was disappeared, and instead, a band of the mutant allele ofapproximately 1.9 kbp in which TaELO2 ORF was substituted for Hygr wasdetected (FIG. 40(B)).

Similarly, a DIG labeled probe that detects TaELO2 was prepared by PCRusing the pair of oligonucleotide primers TaELO2 probe F (30mer: 5′-ATGGCG ACG CGC ACC TCG AAG AGC GCT CCG-3′) (SEQ ID NO: 101) and TaELO2probe R (30mer: 5′-AGG ATC ATC ATG AAC GTG TCG CTC CAG TCG-3′) (SEQ IDNO: 102). [PCR cycles: 98° C. 2 min/98° C. 30 sec, 65° C. 30 sec, 72° C.1 min, 30 cycles/72° C. 7 min/4° C. o]. Southern blotting was performedby the color development method (NBT/BCIP solution) relative to genomeDNA of transformants (clones 1, 8, 9, 10) and a wild-type straindigested with EcoRV. In this case, TaELO2 was detected as a DNA fragmentof approximately 2.5 kbp (FIG. 38(A)).

The result of analysis showed that TaELO2 was detected in the wild-typestrain (FIG. 41, lane 1), whereas in the transformant, TaELO2 was notdetected at all (FIG. 41, lanes 2 to 5).

Furthermore, to verify TaELO2 disruption at an mRNA level, TaELO2 mRNAwas detected by RT-PCR. From the cells of the transformant (clones 1, 8,9, 10) and the wild-type strain on the third day of culturing using a GYliquid culture medium, total RNA was extracted using Sepasol-RNA I Super(available from Nacalai Tesque, Inc.) in the same manner as ComparativeExample 1-1. Then, 50 μg of total RNA cleaned up using RNeasy Mini Kit(available from QIAGEN N.V.) according to the manufacturer's protocolwas treated for 1 hr at 37° C. using 50 U of Recombinant DNase I(available from Takara Bio Inc.), and contaminated genome DNA wasdecomposed and removed. Then, using the obtained total RNA as atemplate, a single-strand cDNA library was prepared according tomanufacturer's instructions using oligo (dT) primer (available fromNovagen Corp.) and PrimeScript Reverse Transcriptase (available fromTakara Bio Inc.). Additionally, using the obtained single-strand cDNAlibrary as a template, TaELO2 ORF was amplified using a pair ofoligonucleotide primers E2 HindIII (described in Comparative Example1-3, SEQ ID NO: 72) and E2 XbaI (described in Comparative Example 1-3,SEQ ID NO: 73) and using LA Taq Hot Start Version (available from TakaraBio Inc.). [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 1 min, 30cycles/68° C. 10 min/4° C. ∞].

The result showed that TaELO2 mRNA was detected in the wild-type strain(FIG. 42, lane 5), whereas TaELO2 mRNA was not detected at all in thetransformant (clones 1, 8, 9, 10) (FIG. 42, lanes 1 to 4).

The above results showed that a TaELO2 deletion homozygote in whichTaELO2 had been completely disrupted was successfully obtained. Theabove results demonstrate that T. aureum ATCC 34304 is diploid.

[Comparative Example 1-12]: Comparison of Fatty Acid Compositions ofWild-Type Strain and TaELO2 Deletion Homozygote

The fatty acid compositions of the TaELO2 deletion homozygote obtainedin Comparative Example 1-11 and the wild-type strain were compared by GCanalysis of the methyl-esterified fatty acids. Specifically, the cellsof the wild-type strain and the TaELO2 deletion homozygote after 5 daysof culturing in a GY liquid culture medium were collected. The fungusbody-derived fatty acids were extracted and methyl-esterified by themethod described in Comparative Example 1-3, and GC analysis wasperformed. In GC analysis, measurement was performed using a gaschromatograph GC-2014 (available from Shimadzu Corporation) under thefollowing conditions. Column: HR-SS-10 (30 m×0.25 mm; available fromShinwa Chemical Industries Ltd.); column temperature: 150° C.→(5°C./min)→220° C. (10 min); carrier gas: He (1.3 mL/min).

As a result, the quantity of EPA serving as a substrate of TaELO2increased up to approximately 2-fold in the TaELO2 deletion homozygote(TaELO2 KO) compared to the wild-type strain (Wild type), and a decreasein the amount of the downstream metabolite DHA was observed (FIG. 43).

As described above, it was confirmed that due to C20 elongase genedisruption in T. aureum ATCC 34304, similar to genus Parietichytriumlabyrinthulids, the quantity of the C20 elongase substrate EPA increasedcompared to the wild-type strain, and conversely, the downstreammetabolite DHA and the like decreased. However, unlike the genusParietichytrium labyrinthulids, the proportion of DHA did not reallydecrease in T. aureum ATCC 34304 even when the C20 elongase gene wasdisrupted. Specifically, the proportion of DHA in the wild-type strainwas 54.38%, whereas the proportion of DHA was 48.77%, which is onlyslightly less in the C20 elongase gene KO strain. A similar trend wasseen for n-6 DPA as well.

As described in Comparative Example 1-2, in T. aureum ATCC 34304, TaELO1is present in addition to TaELO2, which was disrupted this time. InComparative Example 1-3, it was shown that both have Δ5 elongaseactivity (C20 elongase activity). However, it was also clear that the Δ5elongase activity of TaELO1 is considerably lower than the Δ5 elongaseactivity of TaELO2, and the reason that DHA and n-6 DPA did not reallydecrease in the TaELO2 deletion homozygote (TaELO2 KO) is difficult toexplain by the Δ5 elongase activity (=C20 elongase activity) of TaELO1.This suggests the possibility that in Thraustochytrium aureum ATCC34304, DHA and n-6 DPA are produced via another biosynthesis pathway inaddition to the elongase-desaturase pathway.

When such a labyrinthulid is selected, unlike Examples 2, 4, and 6, itis not possible to create a strain which accumulates PUFAs other thanDHA and n-6 DPA even by disruption the C20 elongase gene. Therefore,creating such a strain requires disruption of a gene associated with aDHA and n-6 DPA biosynthesis pathway other than the elongase-desaturasepathway.

Comparative Example 2 Measurement of Fatty Acid Composition of LipidsProduced by PUFA-PKS Gene Disruption and Transformation Strain ofThraustochytrium aureum ATCC 34304 [Comparative Example 2-1]: PUFA-PKSPathway Associated Gene: OrfA Upstream Sequence Cloning

After genome DNA was extracted from Thraustochytrium aureum ATCC 34304by the method described in Example 2-2, A260/280 was measured and theDNA concentration was calculated. Using this extracted genome DNA, agenome cassette library was produced using LA PCR (trade name) in vitroCloning Kit (available from Takara Bio Inc.). A PCR lower primer [RHO20:23mer: 5′-CGA TGA AAG GTC ACA GAA GAG TC-3′ (SEQ ID NO: 103)] was set onthe PUFA-PKS pathway associated gene: OrfA described in Patent Document4, and DNA was amplified by combining with the cassette primerscontained in the above kit. [1st PCR cycles: 98° C. 2 min/98° C. 30 sec,56° C. 30 sec, 72° C. 4 min, 30 cycles/72° C. 5 min]. Then, the 1st PCRamplification product was diluted 100-fold, and the DNA was amplified bycombining the PCR lower primer [RHO20] and the nested primers containedin the above kit. [2nd PCR cycles: 98° C. 2 min/98° C. 30 sec, 56° C. 30sec, 72° C. 4 min, 30 cycles/72° C. 5 min]. The obtained DNA fragmentwas cloned in pGEM-T Easy Vector, and after amplification with E. coli,the sequence was confirmed using a Dye Terminator Cycle Sequencing Kit(available from Beckman Coulter Inc.).

A DNA fragment of 3377 bp (SEQ ID NO: 105) containing 3181 bp (SEQ IDNO: 104) upstream of OrfA was cloned. It became clear that the OrfAupstream DNA sequence information was a total of 3181 bp.

[Comparative Example 2-2]: PUFA-PKS Pathway Associated Gene: OrfADownstream Sequence Cloning

The genome cassette library produced in Comparative Example 2-1 was usedas a template. A PCR upper primer [RHO21: 21mer: 5′-CAG GGC GAG CGA GTGTGG TTC-3′ (SEQ ID NO: 106)] was set on the PUFA-PKS pathway associatedgene: OrfA described in Patent Document 4, and DNA was amplified by themethod described in Comparative Example 2-1. The obtained DNA fragmentwas cloned in pGEM-T Easy Vector, and after amplification with E. coli,the sequence was confirmed using a Dye Terminator Cycle Sequencing Kit(available from Beckman Coulter Inc.). A DNA fragment of 1204 bp (SEQ IDNO: 108) containing 1160 bp (SEQ ID NO: 107) downstream of OrfA wascloned.

A PCR upper primer [RHO28: 20mer: 5′-TGA TGC CGA TGC TAC AAAAG-3′ (SEQID NO: 109)] was again produced on SEQ ID NO: 94, and DNA was amplifiedby the method described in Comparative Example 1-2. The obtained DNAfragment was cloned in pGEM-T Easy Vector, and after amplification withE. coli, the sequence was confirmed using a Dye Terminator CycleSequencing Kit (available from Beckman Coulter Inc.).

Furthermore, a 1488 bp DNA fragment (SEQ ID NO: 110) containing thedownstream sequence was cloned. It became clear that the OrfA downstreamDNA sequence information is a total of 2551 bp (SEQ ID NO: 111).

[Comparative Example 2-3]: Production of PUFA-PKS Pathway AssociatedGene: OrfA Targeting Vector

Using Thraustochytrium aureum ATCC 34304 genome DNA as a template, an18S rDNA sequence (1835 bp, SEQ ID NO: 112) was amplified with PrimeSTARHS DNA Polymerase (available from Takara Bio Inc.). The PCR primers usedwere as shown below. TMO30 was set on the 18S rDNA sequence. TMO31includes the 18S rDNA sequence and the EF1α promoter sequence. [TMO30:30mer: 5′-CGA ATA TTC CTG GTT GAT CCT GCC AGT AGT-3′ (SEQ ID NO: 113),TMO31: 46mer: 5′-GTA ACG GCT TTT TTT GAA TTG CAG GTT CAC TAC GCT TGT TAGAAA C-3′ (SEQ ID NO: 114)]. [PCR cycles: 98° C. 10 sec/98° C. 10 sec,58° C. 30 sec, 72° C. 2 min, 30 cycles/72° C. 2 min].

Furthermore, using Thraustochytrium aureum ATCC 34304 genome DNA as atemplate, an EF1α promoter sequence (661 bp, SEQ ID NO: 115) wasamplified with PrimeSTAR HS DNA Polymerase (available from Takara BioInc.). The PCR primers used were as shown below. TMO32 includes the 18SrDNA sequence and the EF1α promoter sequence. TMO33 includes the EF1αpromoter sequence and an artificially synthesized neomycin resistancegene sequence. [TMO32: 46mer: 5′-GGT TTC CGT AGT GAA CCT GCA ATT CAA AAAAAG CCG TTA CTC ACA T-3′ (SEQ ID NO: 116), TMO33: 46mer: 5′-GCG TGA AGGCCG TCC TGT TCA ATC ATC TAG CCT TCC TTT GCC GCT G-3′ (SEQ ID NO: 117)].[PCR cycles: 98° C. 10 sec/98¹C 10 sec, 58° C. 30 sec, 72° C. 1 min, 30cycles/72° C. 1 min].

Using the artificially synthesized neomycin resistance gene sequence asa template, an artificially synthesized neomycin resistance genesequence (835 bp, SEQ ID NO: 118) was amplified with PrimeSTAR HS DNAPolymerase (available from Takara Bio Inc.). The PCR primers used wereas shown below. TMO34 includes the EF1α promoter sequence and theartificially synthesized neomycin resistance gene sequence. TMO35includes the artificially synthesized neomycin resistance gene sequenceand the EF1α terminator sequence. [TMO34: 45mer: 5′-CAT CGG CAA AGG AAGGCT AGA TGA TTG AAC AGG ACG GCC TTC ACG-3′ (SEQ ID NO: 119), TMO35:46mer: 5′-GCG CAT AGC CGG CGC GGA TCT CAA AAG AAC TCG TCC AGG AGG CGGT-3′ (SEQ ID NO: 120)]. [PCR cycles: 98° C. 10 sec/98° C. 10 sec, 58° C.30 sec, 72° C. 1 min, 30 cycles/72° C. 1 min].

Using Thraustochytrium aureum ATCC 34304 genome DNA as a template, anEF1α terminator sequence (1249 bp, SEQ ID NO: 121) was amplified withPrimeSTAR HS DNA Polymerase (available from Takara Bio Inc.). The PCRprimers used were as shown below. TMO36 includes the artificiallysynthesized neomycin resistance gene sequence and the EF1α terminatorsequence. TMO37 was set within the EF1α terminator. [TMO36: 46mer:5′-TCC TGG ACG AGT TCT TTT GAG ATC CGC GCC GGC TAT GCG CCC GTG C-3′ (SEQID NO: 122), TMO37: 30mer: 5′-CAC TGC AGC GAA AGA CGG GCC GTA AGG ACG-3′(SEQ ID NO: 123)]. [PCR cycles: 98° C. 10 sec/98° C. 10 sec, 58° C. 30sec, 72° C. 2 min, 30 cycles/72° C. 2 min].

Using SEQ ID NOS: 112, 115, 118, and 121 as templates, fusion PCR wasperformed according to the method described in Non-patent Document 9. LATaq Hot Start Version (available from Takara Bio Inc.) was used for theenzymes. In the first amplification, the set of TMO30 (SEQ ID NO: 113)and TMO33 (SEQ ID NO: 117) and the set of TMO34 (SEQ ID NO: 119) andTMO37 (SEQ ID NO: 123) were used. In the second amplification, the setof TMO30 (SEQ ID NO: 113) and TMO37 (SEQ ID NO: 123) was used. Asconditions of the PCR reaction, denaturation was performed at 98° C. for10 sec, and annealing and the elongation reaction were performed whileadjusting as appropriate according to Tm of the primers and the lengthsof the amplification fragments (FIG. 42).

The DNA fragment ligated in this manner (FIG. 44, SEQ ID NO: 124, 4453bp) was cut at the EcoRI site in T. aureum 18S rDNA and at the NcoI sitein the T. aureum EF1α terminator, and was bound to the vector derivedfrom pGEM-T Easy Vector. This was named pRH5 (FIG. 45).

Using Thraustochytrium aureum ATCC 34304 genome DNA as a template, thePCR primers were set in the upstream sequence clarified in ComparativeExample 2-1 (SEQ ID NO: 104) and the PUFA-PKS pathway associated gene:OrfA described in Patent Document 4, and DNA was amplified withPrimeSTAR HS DNA Polymerase with GC Buffer (available from Takara BioInc.). A 1218 bp DNA fragment (SEQ ID NO: 125) was obtained by thisamplification. This was used as the 5′ homologous region of thetargeting vector. The PCR primers used are as shown below. As a linkersequence, an EcoRI site or a HindIII site was appended to each. [RHO33:32mer: 5′-CCC GAA TTC GGA CGA TGA CTG ACT GAC TGA TT-3′ (SEQ ID NO:126), RHO34: 28mer: 5′-CCC AAG CTT GTC TGC CTC GGC TCT TGG T-3′ (SEQ IDNO: 127)]. [PCR cycles: 98° C. 2 min/98° C. 30 sec, 57° C. 30 sec, 72°C. 1 min, 30 cycles/72° C. 3 min].

Using Thraustochytrium aureum ATCC 34304 genome DNA as a template, thePCR primers were set in the downstream sequence clarified in ComparativeExample 2-2 (SEQ ID NO: 111), and DNA was amplified with PrimeSTAR HSDNA Polymerase with GC Buffer (available from Takara Bio Inc.). Δ1000 bpDNA fragment (SEQ ID NO: 128) was obtained by this amplification. Thiswas used as the 3′ homologous region of the targeting vector. The PCRprimers used were as shown below. An NcoI site as a linker sequence wasappended to each. [RHO29: 28mer: 5′-CCC CCA TGG TGT TGC TGT GGG ATT GGTC-3′ (SEQ ID NO: 129), RHO30: 30mer: 5′-CCC CCA TGG CTC GGT TAC ATC TCTGAG GAA-3′ (SEQ ID NO: 130)]. [PCR cycles: 98° C. 2 min/98° C. 30 sec,57° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 3 min].

The amplified upstream sequence was ligated to the EcoRI site and theHindIII site in pRH5 illustrated in FIG. 43. The amplified downstreamsequence was ligated to the NcoI site. This vector was named pRH21.

The targeting vector (pRH21) obtained using the produced artificiallysynthesized neomycin resistance gene is illustrated in FIG. 44.

[Comparative Example 2-4]: Production of PUFA-PKS Pathway AssociatedGene: OrfA Targeting Vector (Hygromycin Resistance Gene)

Using pRH32 (FIG. 6) described in Example 2-3 as a template, a ubiquitinpromoter—hygromycin resistance gene fragment (1632 bp, SEQ ID NO: 131)was amplified with PrimeSTAR HS DNA Polymerase with GC Buffer (availablefrom Takara Bio Inc.). The PCR primers used were as shown below. RHO59was set on the ubiquitin promoter, and a HindIII site was appended as alinker sequence. RHO60 contains a stop codon of the hygromycinresistance gene sequence, and has the linker sequences SphI and SalI.[RHO59: 36mer: 5′-CCC AAG CTT GCC GCA GCG CCT GGT GCA CCC GCC GGG-3′(SEQ ID NO: 132), RHO60: 43mer: 5′-CCC GCA TGC GTC GAC TAT TCC TTT GCCCTC GGA CGA GTG CTG G-3′ (SEQ ID NO: 133)]. [PCR cycles: 98° C. 2min/98° C. 30 sec. 68° C. 2 min. 30 cycles/68° C. 2 min].

The amplified fragment was ligated to the HindIII and SphI sites ofpRH21 (FIG. 46) described in Comparative Example 2-3 (FIG. 47, pRH30).

Using Thraustochytrium aureum ATCC 34304 genome DNA as a template, theDNA was amplified with PrimeSTAR HS DNA Polymerase with GC Buffer(available from Takara Bio Inc.) using the produced PCR primers in thedownstream sequence (SEQ ID NO: 111) clarified in Comparative Example2-2. A 1000 bp DNA fragment (SEQ ID NO: 134) was obtained by thisamplification. This was used as the 3′ homologous region of thetargeting vector. The PCR primers used were as shown below. A SalI siteas a linker sequence was also appended. [RHO61: 29mer: 5′-CCC GTC GACGTG TTG CTG TGG GAT TGG TC-3′ (SEQ ID NO: 135), RHO62: 29mer: 5′-CCC GTCGAC TCG GTT ACA TCT CTG AGG AA-3′ (SEQ ID NO: 136)]. [PCR cycles: 98° C.2 min/98° C. 30 sec, 57° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 3min].

The amplified downstream sequence was ligated to the SalI site of pRH30(FIG. 45). This was named pRH33. The targeting vector (pRH33) obtainedusing the produced hygromycin resistance gene is illustrated in FIG. 48.

[Comparative Example 2-5]: PUFA-PKS Pathway Associated Gene: OrfATargeting Vector Transfer

Using the targeting vectors produced in Comparative Examples 2-3 and 2-4as templates, the genes were amplified with PrimeSTAR Max DNA Polymerase(available from Takara Bio Inc.) using RHO30 (described in ComparativeExample 2-3, SEQ ID NO: 130) and RHO33 (described in Comparative Example2-3, SEQ ID NO: 126) as primers. [PCR cycles: 98° C. 2 min/98° C. 30sec, 60° C. 30 sec, 72° C. 1 min, 30 cycles/72° C. 3 min]. After phenolchloroform extraction and chloroform extraction, the DNA underwentethanol precipitation, and the precipitate was dissolved in 0.1×TE.A260/280 was measured and the DNA concentration was calculated. Thetransfer fragment obtained when pRH21 (FIG. 46) described in ComparativeExample 2-3 was used as a template was 3705 bp, and resulted in asequence including Thraustochytrium aureum OrfA gene upstream—EF1αpromoter sequence—artificially synthesized neomycin resistance genesequence—Thraustochytrium aureum OrfA gene downstream (SEQ ID NO: 137).The transfer fragment obtained when pRH33 (FIG. 46) described inComparative Example 2-4 was used as a template was 3826 bp, and resultedin a sequence including Thraustochytrium aureum OrfA geneupstream—ubiquitin promoter sequence—hygromycin resistance genesequence—Thraustochytrium aureum OrfA gene downstream (SEQ ID NO: 138).

The Thraustochytrium aureum ATCC 34304 strain was cultured for 4 days ina GY culture medium, and cells in the logarithmic growth phase were usedin gene transfer. To cells corresponding to OD600=1 to 1.5, 0.625 μg ofDNA fragment was transformed by the gene gun method (microcarrier: 0.6micron gold particles, target distance: 6 cm, chamber vacuum: 26 mmHg,rupture disk: 1100 psi). After a recovery time of 4 to 6 hr, thetransgenic cells were spread on a PDA agar plate culture medium(containing 2 mg/mL of G418 or containing 2 mg/mL of hygromycin). As aresult, from 100 to 200 cells of drug resistant strain per shot wereobtained.

[Comparative Example 2-6]: Identification of PUFA-PKS Pathway AssociatedGene: OrfA Gene Targeting Homologous Recombinant

After genome DNA was extracted from Thraustochytrium aureum ATCC 34304and a hetero homologous recombinant and homo homologous recombinant (PKSpathway associated gene disruption strain) by the method described inExample 2-2, A260/280 was measured and the DNA concentration wascalculated.

After the genome DNA was cut with a restriction enzyme, the obtainedgenome DNA underwent electrophoresis in approximately 2 to 3 μg per wellof 0.7% SeaKem GTG agarose gel (available from Takara Bio Inc.). Thiswas transformed to a nylon membrane, and hybridized at 54° C. for 16 hrwith a probe produced using DIG System (available from Roche AppliedScience, Inc.).

The primers used in probe production were as follows. 5′ side [RHO37:22mer: 5′-GAA GCG TCC CGT AGA TGT GGT C-3′ (SEQ ID NO: 139), RHO38:21mer: 5′-GCC CGA GAG GTC AAA GTA CGC-3′ (SEQ ID NO: 140)]; 3′ side[RHO39: 20mer: 5′-GCG AGC CCA GGT CCA CTT GC-3′ (SEQ ID NO: 141). RHO40:22mer: 5′-CAG CCC GAT GAA AAA CTT GGT C-3′ (SEQ ID NO: 142)]. [PCRcycles: 98° C. 2 min/98° C. 30 sec, 60° C. 30 sec, 72° C. 2 min, 30cycles/72° C. 3 min]. The positions of the restriction enzymes and theprobes used are illustrated in FIG. 49. The hybridized probes weredetected using the color development method (NBT/BCIP solution).

In analysis of both the 5′ side and the 3′ side, bands were observed atthe expected sizes when the drug resistance genes caused homologousrecombination (FIG. 50).

Comparative Example 2-7

Thraustochytrium aureum ATCC 34304 and the gene disruption strain werecultured according to the method described in Example 2-9, and afterfreeze drying, the fatty acids were methyl-esterified and analyzed usingGC.

The changes in the fatty acid composition are shown in FIG. 51. FIG. 52shows the proportion when the wild-type strain is taken as 100%. FIG. 52shows that, of the total fatty acid composition, ARA is 3.10%, DGLA is0.23%, ETA is 0.04%, EPA is 6.82%, n-6 DPA is 10.66%, and DHA is 22.58%.FIG. 52 shows that, by GC area, LA/DHA is 0.05, GLA/DHA is 0.03, DGLADHAis 0.01, ARA/DHA is 0.1, EPA/DHA is 0.3, LA/EPA is 0.16, GLA/EPA is0.11, DTA/EPA is 0.29, DTA/ARA is 0.65, DTA/DGLA is 8.7, LA/n-6 DPA is0.1, GLA/n-6 DPA is 0.07, DGLA/n-6 DPA is 0.02, ARA/n-6 DPA is 0.3,EPA/n-6 DPA is 0.6, DGLA/LA is 0.2, ARA/LA is 2.9, EPA/LA is 6.4, DTA/LAis 1.9, DGLA/GLA is 0.3, ARA/GLA is 4.0, n-6 DPA/DTA is 5.3. DHA/n-3 DPAis 20.0, C20 PUFA/C22 PUFA is 0.3, and n-6 PUFA/n-3 PUFA is 0.52.

As a result, when the PUFA-PKS pathway associated gene: OrfA wasdisrupted in Thraustochytrium aureum, DPA (C22:5n-6) exhibited anincreasing trend and DHA (C22:6n-3) exhibited a decreasing trend.

It is known that in the genus Schizochytrium and genus Aurantiochytrium,exogenous PUFAs become necessary when a PUFA-PKS pathway gene isdisrupted, and breeding is not possible unless exogenous PUFAs aresupplied (Non-patent Document 4). Unlike these organisms, however,Thraustochytrium aureum ATCC 34304 can be cultured without supplementingthe culture medium with exogenous PUFAs when a PUFA-PKS pathwayassociated gene is disrupted. Furthermore, disruption of a PUFA-PKSpathway associated gene, decreased DHA only to approximately ⅔ andslightly increased DPA (C22:5n-6) compared to the wild-type strain.

The above results suggest the possibility that DHA and n-6 DPA areproduced via another biosynthesis pathway in addition to the PUFA-PKSpathway in Thraustochytrium aureum ATCC 34304. The reason thatThraustochytrium aureum ATCC 34304 can be cultured without supplementingthe culture medium with exogenous PUFAs is surmised to be thatendogenous PUFAs are supplied via a biosynthesis pathway other than thePUFA-PKS pathway.

Comparative Example 3 Measurement of Fatty Acid Composition of LipidsProduced by PUFA-PKS Gene and C20 Elongase Gene Disruption andTransformation Strain of Thraustochytrium aureum ATCC 34304 [ComparativeExample 3-1]: Cloning of Upstream Sequence of Thraustochytrium aureumC20 Elongase Gene

The genome cassette library produced in Comparative Example 2-1 was usedas a template. A PCR lower primer [RHO71: 22mer: 5′-GGG AGC GCA GGG AAAACG GTC T-3′ (SEQ ID NO: 143)] was produced on the C20 elongase geneupstream sequence (SEQ ID NO: 76) described in Comparative Example 1-4,and the gene was amplified by combining with the cassette primerscontained in the kit described in Comparative Example 2-1. [1st PCRcycles: 98° C. 2 min/98° C. 30 sec, 56° C. 30 sec, 72° C. 4 min, 30cycles/72° C. 5 min]. Then, the 1st PCR amplification product wasdiluted 100-fold, and the gene was amplified by combining a PCR lowerprimer [RHO72: 20mer: 5′-CCA GCC CAC GTC GTC GGA GC-3′ (SEQ ID NO: 144)]and the nested primers contained in the kit described in ComparativeExample 2-1. [2nd PCR cycles: 98° C. 2 min/98° C. 30 sec, 56° C. 30 sec,72° C. 4 min, 30 cycles/72° C. 5 min]. The obtained DNA fragment wascloned in pGEM-T Easy Vector, and after amplification with E. coli, thesequence was confirmed using a Dye Terminator Cycle Sequencing Kit(available from Beckman Coulter Inc.).

A 2297 bp DNA fragment (SEQ ID NO: 145) containing the 3277 bp to 981 bpregion upstream of the C20 elongase gene was cloned.

[Comparative Example 3-2]: Cloning of Downstream Sequence of C20Elongase Gene

The genome cassette library produced in Comparative Example 2-1 was usedas a template. A PCR upper primer [RHO87: 23mer: 5′-GCC GCT CAT GCC CACGCT CAA AC-3′ (SEQ ID NO: 146)] was produced on the C20 elongase genedownstream sequence (SEQ ID NO: 77) described in Comparative Example1-4, and the gene was amplified by combining with the cassette primerscontained in the kit described in Comparative Example 2-1. [1st PCRcycles: 98° C. 2 min/98° C. 30 sec, 56° C. 30 sec, 72° C. 4 min, 30cycles/72° C. 5 min]. Then, the 1st PCR amplification product wasdiluted 100-fold, and the gene was amplified by combining a PCR lowerprimer [RHO73: 23mer: 5′-CTT TCG GCT GCC AGG AAT CTA CG-3′ (SEQ ID NO:147)] and the nested primers contained in the kit described inComparative Example 2-1. [2nd PCR cycles: 98° C. 2 min/98° C. 30 sec,56° C. 30 sec, 72° C. 4 min, 30 cycles/72° C. 5 min]. The obtained DNAfragment was cloned in pGEM-T Easy Vector, and after amplification withE. coli, the sequence was confirmed using a Dye Terminator CycleSequencing Kit (available from Beckman Coulter Inc.).

A 2189 bp DNA fragment (SEQ ID NO: 148) containing the 1106 bp to 3294bp region downstream of the C20 elongase gene was cloned.

[Comparative Example 3-3]: Production of Blasticidin Resistance GeneCassette

Using genome DNA from Thraustochytrium aureum ATCC 34304 as a template,an ubiquitin promoter sequence (618 bp, SEQ ID NO: 149) was amplifiedwith PrimeSTAR HS DNA Polymerase with GC Buffer (available from TakaraBio Inc.). The PCR primers used were as shown below. RHO53 was set onthe ubiquitin promoter sequence, and includes a BglII linker sequence(Example 2-2. SEQ ID NO: 5). RHO48 includes the ubiquitin promotersequence and a blasticidin resistance gene sequence. [RHO48: 58mer:5′-CTT CTT GAG ACA AAG GCT TGG CCA TGT TGG CTA GTG TTG CTT AGG TCG CTTGCT GCT G-3′) (SEQ ID NO: 150)]. [PCR cycles: 98° C. 2 min/98° C. 10sec, 68° C. 1 min, 30 cycles/68° C. 1 min].

Using pTracer-CMV/Bsd/lacZ (available from Invitrogen Corp.) as atemplate, a blasticidin resistance gene (432 bp, SEQ ID NO: 151) wasamplified with PrimeSTAR HS DNA Polymerase with GC Buffer. The PCRprimers used were as shown below. RHO47 includes the ubiquitin promotersequence and the blasticidin resistance gene sequence. RHO49 includesthe blasticidin resistance gene sequence and has a BglII linkersequence. [RHO47: 54mer: 5′-AGC GAC CTA AGC AAC ACT AGC CAA CAT GGC CAAGCC TTT GTC TCA AGA AGA ATC-3′ (SEQ ID NO: 152), RHO49: 38mer: 5′-CCCAGA TCT TAG CCC TCC CAC ACA TAA CCA GAG GGC AG-3′ (SEQ ID NO: 153)].[PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 1 min, 30 cycles/68° C.1 min].

Using SEQ ID NOS: 149 and 151 as templates, fusion PCR was performedusing RHO53 (described in Example 2-2, SEQ ID NO: 5) and RHO49 (SEQ IDNO: 153) according to the method described in Non-patent Document 9.Amplification was performed using LA Taq Hot Start Version (availablefrom Takara Bio Inc.) as the enzyme under the following conditions, andthen the amplified product was digested with BglII. [PCR cycles: 94° C.2 min/94° C. 20 sec. 55° C. 30 sec. 68° C. 1 min, 30 cycles/68° C. 1 min(1° C./10 sec from 55° C. to 68° C.)]. (FIG. 53).

The Thraustochytrium aureum ATCC 34304-derived ubiquitinpromoter—pTracer-CMV/Bsd/lacZ-derived blasticidin resistance genesequence (1000 bp, SEQ ID NO: 154) fused as described above was digestedwith BglII, and the resultant was bound to the BamHI site of pRH27 (FIG.2) described in Example 2-1. After amplification of the produced plasmidwith E. coli, the sequence was confirmed using a Dye Terminator CycleSequencing Kit (available from Beckman Coulter Inc.). This was namedpRH38.

The produced blasticidin resistance gene cassette (pRH38) is illustratedin FIG. 54.

[Comparative Example 3-4]: Production of GFP Fusion Zeocin ResistanceGene Cassette

Using genome DNA from Thraustochytrium aureum ATCC 34304 as a template,an ubiquitin promoter sequence (812 bp, SEQ ID NO: 155) was amplifiedwith PrimeSTAR HS DNA Polymerase with GC Buffer (available from TakaraBio Inc.). The PCR primers used were as shown below. TMO38 was set onthe ubiquitin promoter sequence. TMO39 includes the ubiquitin promotersequence and an enhanced GFP gene sequence. [TMO38: 29mer: 5′-TCG GTACCC GTT AGA ACG CGT AAT ACG AC-3′ (SEQ ID NO: 156), TMO39: 41mer: 5′-TCCTCG CCC TTG CTC ACC ATG TTG GCT AGT GTT GCT TAG GT-3′ (SEQ ID NO: 157)].[PCR cycles: 98° C. 10 sec/98° C. 10 sec, 58° C. 30 sec, 72° C. 1 min,30 cycles/72° C. 1 min].

Using an enhanced GFP gene sequence (available from ClontechLaboratories, Inc.) as a template, the enhanced GFP gene sequence (748bp, SEQ ID NO: 158) was amplified with PrimeSTAR HS DNA Polymerase(available from Takara Bio Inc.). The PCR primers used were as shownbelow. TMO40 includes the ubiquitin promoter sequence and the enhancedGFP gene sequence. RHO91 includes the enhanced GFP sequence and a zeocinresistance gene sequence. [TMO40: 41mer: 5′-ACC TAA GCA ACA CTA GCC AACATG GTG AGC AAG GGC GAG GA-3′ (SEQ ID NO: 159), RHO91: 58mer: 5′-GAA CGGCAC TGG TCA ACT TGG CGT CCA TGC CGA GAG TGA TCC CGG CGG CGG TCA CGA A-3′(SEQ ID NO: 160)]. [PCR cycles: 98° C. 10 sec/98° C. 10 sec, 58° C. 30sec, 72° C. 2 min, 30 cycles/72° C. 2 min].

Using SEQ ID NOS: 156 and 158 as templates, fusion PCR was performed byLA Taq Hot Start Version (available from Takara Bio Inc.) according tothe method described in Non-patent Document 9. TMO38 (SEQ ID NO: 156)and RHO91 (SEQ ID NO: 160) were used as primers, and the conditions wereas follows: PCR cycles: 94° C. 2 min/94° C. 20 sec, 55° C. 30 sec, 68°C. 2 min, 30 cycles/68° C. 2 min (1° C./10 sec from 55° C. to 68° C.)(FIG. 55, 1519 bp, SEQ ID NO: 161).

Using SEQ ID NO: 161 as a template, the ubiquitin promotersequence—enhanced GFP gene sequence (1319 bp, SEQ ID NO: 162) wasamplified with PrimeSTAR HS DNA Polymerase (available from Takara BioInc.). The primers used were as follows. RHO53 (Example 2-2, SEQ ID NO:5) contains the ubiquitin promoter sequence and has a BglII site. RHO91(SEQ ID NO: 160) includes the enhanced GFP sequence and a zeocinresistance gene sequence. [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68°C. 2 min, 30 cycles/68° C. 2 min].

Using pcDNA 3.1 Zeo(+) as a template, a zeocin resistance gene sequence(408 bp, SEQ ID NO: 163) was amplified with PrimeSTAR HS DNA Polymerase(available from Takara Bio Inc.). RHO92 includes the enhanced GFPsequence and a zeocin resistance gene sequence. RHO64 contains thezeocin resistance gene sequence and has a BglII site. [RHO92: 54mer:5′-CGC CGC CGG GAT CAC TCT CGG CAT GGA CGC CAA GTT GAC CAG TGC CGT TCCGGT-3′ (SEQ ID NO: 164), RHO64: 38mer: 5′-CCC AGA TCT CAG TCC TGC TCCTCG GCC ACG AAG TGC AC-3′ (SEQ ID NO: 165)]. [PCR cycles: 98° C. 2min/98° C. 10 sec, 68° ° C. 1 min, 30 cycles/68° C. 1 min].

Using SEQ ID NOS: 162 and 163 as templates, fusion PCR was performed byLA Taq Hot Start Version (available from Takara Bio Inc.) according tothe method described in Non-patent Document 9. RHO53 (Example 2-2, SEQID NO: 5) and RHO64 (SEQ ID NO: 165) were used as primers, and theconditions were as follows: PCR cycles: 94′C 2 min/94° C. 20 sec, 68° C.2 min, 30 cycles/68° C. 2 min (l C/10 sec from 55° C. to 68° C.) (FIG.56).

The Thraustochytrium aureum ATCC 34304-derived ubiquitinpromoter—enhanced GFP gene—pcDNA 3.1 Zeo(+)-derived zeocin resistancegene (FIG. 56, 1677 bp, SEQ ID NO: 166) fused as described above wasdigested with BglII, and the resultant was bound to the BamHI site ofpRH27 (FIG. 2) described in Example 2-1. After amplification of theproduced plasmid with E. coli, the sequence was confirmed using a DyeTerminator Cycle Sequencing Kit (available from Beckman Coulter Inc.).This was named pRH51.

The produced GFP fusion zeocin resistance gene cassette (pRH51) isillustrated in FIG. 57.

[Comparative Example 3-5]: Production of Plasmid Serving as Base forProduction of C20 Elongase Gene Targeting Vector

Using Thraustochytrium aureum ATCC 34304 genome DNA as a template, a C20elongase gene and its surrounding sequence were amplified with PCR byPrimeSTAR HS DNA Polymerase (available from Takara Bio Inc.) (2884 bp,SEQ ID NO: 167). The PCR primers used were as follows. Both primerscontained an EcoI linker sequence. KSO9 was set in the C20 elongase geneupstream (SEQ ID NO: 76), and KSO10 was set in the C20 elongase genedownstream (SEQ ID NO: 77). [KSO9: 50mer: 5′-CCC GAA TTC ACT AGT GAT TCTCCC GGG TGG ACC TAG CGC GTG TGT CAC CT-3′ (SEQ ID NO: 168), KSO10:40mer: 5′-CCC GAA TTC GAT TAT CCC GGG GCC GAG AAC GGG GTC GCC C-3′ (SEQID NO: 169)]. [PCR cycles: 98° C. 2 min/98° C. 10 sec, 68° C. 3.5 min,30 cycles/68° C. 10 min]. PrimeSTAR HS DNA Polymerase (available fromTakara Bio Inc.) was used for the enzymes, and after amplification, theamplified products were digested with EcoRI, and then cloned at thevector EcoRI site of pBlueScript (SK) (available from Stratagene Corp.).After amplification with E. coli, the sequence was confirmed using a DyeTerminator Cycle Sequencing Kit (available from Beckman Coulter Inc.)(FIG. 58).

A primer set that was set up in the reverse direction with the objectiveof deleting a portion of the C20 elongase gene sequence and inserting aBglII site (1939 bp, SEQ ID NO: 170) was prepared using the plasmidillustrated in FIG. 58 as a template, and the set was amplified withPrimeSTAR Max DNA Polymerase (available from Takara Bio Inc.). The PCRprimers used were as shown below, both of which have a BgII linkersequence. [RHO69: 38mer: 5′-CCC AGA TCT ACC TGT TTC CGG CTG GCT CCC GAGCCA TG-3′ (SEQ ID NO: 171), RHO70: 38mer: 5′-CCC AGA TCT GGT CGC GTT TACAAA GCA GCG CAG CAA CA-3′ (SEQ ID NO: 172)]. [PCR cycles: 98° C. 2min/98° C. 10 sec, 68° C. 1.5 min, 30 cycles/68° C. 1.5 min]. Afteramplification under the above conditions, the amplified product wasdigested with BglII and then self-ligated. After the ligated sample wasamplified with E. coli, the sequence was confirmed using a DyeTerminator Cycle Sequencing Kit (available from Beckman Coulter Inc.).This was named pRH40.

The produced plasmid (pRH40) serving as a base for production of a C20elongase gene targeting vector is illustrated in FIG. 59.

[Comparative Example 3-6]: Production of Targeting Vectors (BlasticidinResistance Gene and GFP Fusion Zeocin Resistance Gene)

pRH38 (FIG. 52) described in Comparative Example 3-3 was digested withBglII, and a DNA fragment containing a blasticidin resistance genecassette was bound to the BglII site of pRH40 (FIG. 59) described inComparative Example 3-5. This was named pRH43.

pRH51 (FIG. 55) described in Comparative Example 3-4 was digested withBglII, and a DNA fragment containing a GFP fusion zeocin resistance genecassette was bound to the BglII site of pRH40 (FIG. 57) described inComparative Example 3-5. This was named pRH54.

The two produced targeting vectors (pRH43 and 54) are illustrated inFIG. 60.

[Comparative Example 3-7]: Transfer of C20 Elongase Gene TargetingVectors into Thraustochytrium aureum OrfA Disruption Strain

Using the two targeting vectors produced in Comparative Example 3-6 astemplates, the genes were amplified with PrimeSTAR Max DNA Polymerase(available from Takara Bio Inc.) using KSO11 and KSO12 as primers. KSO11was set upstream of the Thraustochytrium aureum C20 elongase gene, andKSO12 was set downstream of the Thraustochytrium aureum C20 elongasegene. [KSO111: 31mer: 5′-CTC CCG GGT GGA CCT AGC GCG TGT GTC ACC T-3′(SEQ ID NO: 173), KSO12: 27mer: 5′-ATC CCG GGG CCG AGA ACG CCC TCGCCC-3′ (SEQ ID NO: 174)]. [PCR cycles: 98° C. 2 min/98° C. 30 sec, 68°C. 2 min, 30 cycles/68° C. 2 min]. After phenol chloroform extractionand chloroform extraction, the DNA underwent ethanol precipitation, andthe precipitate was dissolved in 0.1×TE. A260/280 was measured and theDNA concentration was calculated. The transfer fragment obtained whenpRH43 (FIG. 60) described in Comparative Example 3-6 was used as atemplate was 3215 bp, and resulted in a sequence includingThraustochytrium aureum C20 elongase gene upstream—ubiquitinpromoter—blasticidin resistance gene sequence—SV40 terminatorsequence—Thraustochytrium aureum C20 elongase gene downstream (SEQ IDNO: 175). The transfer fragment obtained when pRH54 (FIG. 60) describedin Comparative Example 3-6 was used as a template was 3887 bp, andresulted in a sequence including Thraustochytrium aureum C20 elongasegene upstream—ubiquitin promoter—enhanced GFP gene sequence—zeocinresistance gene sequence—SV40 terminator sequence—Thraustochytriumaureum C20 elongase gene downstream (SEQ ID NO: 176).

The PUFA-PKS pathway associated gene: OrfA gene disruption straindescribed in Comparative Example 2 was cultured for 4 days in a GYculture medium, and cells in the logarithmic growth phase were used ingene transfer. To cells corresponding to OD600=1 to 1.5, 0.625 μg of DNAfragment was transformed by the gene gun method (microcarrier: 0.6micron gold particles, target distance: 6 cm, chamber vacuum: 26 mmHg,rupture disk: 1100 psi). After a recovery time of 4 to 6 hr, thetransgenic cells were spread on a PDA agar plate culture medium(containing 2 mg/mL of G418 or containing 2 mg/mL of hygromycin). As aresult, from 100 to 200 cells of drug resistant strain per shot wereobtained.

[Comparative Example 3-8]: Identification of C20 Elongase Gene TargetingHomologous Recombinant

After genome DNA was extracted from the C20 elongase gene disruptionstrain in the Thraustochytrium aureum OrfA disruption strain andThraustochytrium aureum by the method described in Example 2-2,A260/A280 was measured and the DNA concentration was calculated.

After the genome DNA was cut with a restriction enzyme, and underwentelectrophoresis in approximately 2 to 3 μg per well of 0.7% SeaKem GTGagarose gel (available from Takara Bio Inc.). This was transformed to anylon membrane, and hybridized for at 51° C. 16 hr with a probe producedusing DIG System (available from Roche Applied Science, Inc.). Theprimers used in probe production were as follows. 5′ side [RHO94: 21mer:5′-ACG TCC GCT TCA AAC ACC TCG-3′ (SEQ ID NO: 177), RHO95: 24mer: 5′-TCGGAA CAA CTG GAA CAA CTA AAG-3′ (SEQ ID NO: 178)]; 3′ side [RHO96: 22mer:5′-ATG TCG CTC TCC TTC TTC TCA G-3′ (SEQ ID NO: 179), RHO97: 21mer:5′-TCG GCT CCT GGA AAG TGC TCT-3′ (SEQ ID NO: 180)]. [PCR cycles: 98° C.2 min/98° C. 30 sec, 58° C. 30 sec. 72° C. 1 min, 30 cycles/72° C. 3min]. The positions of the restriction enzymes and the probes used areillustrated in FIG. 61. The hybridized probes were detected using thecolor development method (NBT/BCIP solution).

In analysis of both the 5′ side and the 3′ side, bands were observed atthe expected sizes when the drug resistance genes caused homologousrecombination (FIG. 62). The experiment reveals that theThraustochytrium aureum ATCC 34304 strain does not require nutrientseven when the PKS pathway associated gene: OrfA and the C20 elongasegene are deleted.

[Comparative Example 3-9]: Change in Fatty Acid Composition by C20Elongase Gene Disruption in Thraustochytrium aureum OrfA DisruptionStrain

Thraustochytrium aureum ATCC 34304 and the gene disruption strain werecultured according to the method described in Example 2-9, and afterfreeze drying, the fatty acids were methyl-esterified and analyzed usingGC. In GC analysis, measurement was performed using a gas chromatographGC-2014 (available from Shimadzu Corporation) under the followingconditions. Column: HR-SS-10 (30 m×0.25 mm; available from ShinwaChemical Industries Ltd.); column temperature: 150° C.→(5° C./min)→220°C. (10 min); carrier gas: He (1.3 mL/min).

The changes in the fatty acid composition are shown in FIG. 63.Furthermore, FIG. 64 shows the proportion when the wild-type strain istaken as 100%. FIG. 64 shows that, of the total fatty acid composition,ARA is 19.50%, DGLA is 1.81%, ETA is 0.31%, EPA is 24.92%, n-6 DPA is5.90%, and DHA is 6.78%. FIG. 64 shows that, by GC area, LA/DHA is 0.25,GLA/DHA is 0.07, DGLADHA is 0.27, ARA/DHA is 2.88, EPA/DHA is 3.68,LA/EPA is 0.07, GLA/EPA is 0.02, DTA/EPA is 0.02, DTA/ARA is 0.02,DTA/DGLA is 0.26, LA/n-6 DPA is 0.29, GLA/n-6 DPA is 0.08, DGLA/n-6 DPAis 0.31, ARA/n-6 DPA is 3.31, EPA/n-6 DPA is 4.22, DGLA/LA is 1.06,ARA/LA is 11.40, EPA/LA is 14.57, DTA/LA is 0.27, DGLA/GLA is 4.02,ARA/GLA is 43.33, n-6 DPA/DTA is 12.55, DHA/n-3 DPA is 11.69, C20PUFA/C22 PUFA is 3.39, and n-6 PUFA/n-3 PUFA is 0.85.

As a result, when the C20 elongase gene was disrupted in theThraustochytrium aureum OrfA disruption strain, C20:4n-6 (ARA) increasedapproximately 8-fold, C20:5n-3 (EPA) increased approximately 4-fold, andC22:6n-3 (DHA) decreased to approximately ⅕.

Thus, it was demonstrated that in order to create a strain in which theproduced quantity of DHA and DPA n-6 are markedly reduced fromThraustochytrium aureum ATCC 34304, which has both an endogenouselongase-desaturase pathway and an endogenous PUFA-PKS pathway, both agene of an enzyme constituting the elongase-desaturase pathway (forexample, the C20 elongase gene) and a PUFA-PKS pathway associated geneneed to be disrupted.

Comparative Example 4 Measurement of Fatty Acid Composition of LipidsProduced by PUFA-PKS Gene and Δ4 Desaturase Gene Disruption andTransformation Strain of Thraustochytrium aureum ATCC 34304 [ComparativeExample 4-1]: Cloning Sequence of 1071 bp Upstream of Δ4 Desaturase Geneto 1500 bp within Δ4 Desaturase Gene of Thraustochytrium Aureum ATCC34304 Strain

Genome DNA of the Thraustochytrium aureum ATCC 34304 strain extracted bythe method described in Example 2-2 was read, and a gene sequence havinghigh homology to known Δ4 desaturase was searched for. Two PCR primerswere designed based on the search results. TMO3 is a sequence located at1071 to 1049 bp upstream of the Δ4 desaturase gene of theThraustochytrium aureum ATCC 34304 strain, and TMO4 is a sequence withinthe protein coding region located at 1477 to 1500 bp counting from thestart codon. [TMO3: 23mer: 5′-GGC GGA GCG AAG TGT GAA AGT TA-3′ (SEQ IDNO: 181), TMO4: 24mer: 5′-GCG ACA GCA TCT TGA AAT AGG CAG-3′ (SEQ ID NO:182)]. Using genome DNA of the Thraustochytrium aureum ATCC 34304 strainas a primer, the sequence of 1071 bp upstream of the Δ4 desaturase geneto 1500 bp within the Δ4 desaturase gene (2571 bp, SEQ ID NO: 183) ofThraustochytrium aureum ATCC 34304 strain was amplified using these twoprimers. The amplification conditions were as follows. [PCR cycles: 98°C. 2 min/98° C. 20 sec, 60° C. 30 sec, 72° C. 3 min, 30 cycles/72° C. 8min]. The obtained DNA fragment was cloned in pGEM-T Easy Vector, andafter amplification with E. coli, the sequence was confirmed using a DyeTerminator Cycle Sequencing Kit (available from Beckman Coulter Inc.).This was named pTM1 (FIG. 65).

[Comparative Example 4-2]: Production of Plasmid Serving as Base forProduction of Δ4 Desaturase Gene Targeting Vector

Using pTM1 (FIG. 65) produced in Comparative Example 4-1 as a template,a primer set set in the reverse direction so as to delete a 556 bpsequence (616 bp, SEQ ID NO: 184) containing 60 bp upstream of the Δ4desaturase gene and the start codon within the Δ4 desaturase gene and toproduce a BglII site in the deleted portion was prepared. TMO7 and TMO8both contain a BglII sequence. PrimeSTAR Max DNA Polymerase (availablefrom Takara Bio Inc.) was used in amplification. [TMO7: 25mer: 5′-CAGGAG ATC TCC AAG TCG CGA TTC A-3′ (SEQ ID NO: 185), TMO8: 26mer: 5′-CTTGGA GAT CTC CTG CCC GTC CCG AA-3′ (SEQ ID NO: 186)]. [PCR cycles: 98° C.3 min/98° C. 10 sec, 55° C. 15 sec, 72° C. 30 sec, 30 cycles/72° C. 30sec]. After amplification under the above conditions, the amplifiedproduct was purified by electrophoresis using agarose gel. Aftertransforming the obtained DNA fragment into E. coli, and amplifying, thesequence was confirmed using a Dye Terminator Cycle Sequencing Kit(available from Beckman Coulter Inc.). This was named pTM2.

The produced plasmid (pTM2) serving as a base for production of a Δ4desaturase gene targeting vector is illustrated in FIG. 66.

[Comparative Example 4-3]: Production of Targeting Vectors (BlasticidinResistance Gene and GFP Fusion Zeocin Resistance Gene)

pRH38 (FIG. 54) described in Comparative Example 3-3 was digested withBglII, and a DNA fragment containing a blasticidin resistance genecassette was bound to the BglII site of pTM2 (FIG. 66) described inComparative Example 4-2. This was named pTM6, pRH51 (FIG. 57) describedin Comparative Example 3-4 was digested with BglII, and a DNA fragmentcontaining a GFP fusion zeocin resistance gene cassette was bound to theBglII site of pTM2 (FIG. 66) described in Comparative Example 4-2. Thiswas named pTM8.

The two produced targeting vectors (pTM6 and 8) are illustrated in FIG.67.

[Comparative Example 4-4]: Transfer of Δ4 Desaturase Gene TargetingVectors into Thraustochytrium aureum OrfA Disruption Strain

Using the two targeting vectors produced in Comparative Example 4-3 astemplates, the genes were amplified with PrimeSTAR Max DNA Polymerase(available from Takara Bio Inc.) using TMO3 (described in ComparativeExample 4-1, SEQ ID NO: 181) and TMO4 (described in Comparative Example4-1, SEQ ID NO: 182) as primers. [PCR cycles: 98° C. 3 min/98° C. 10sec, 55° C. 5 sec, 72° C. 4 min, 30 cycles/72° C. 3 min]. After phenolchloroform extraction and chloroform extraction, the DNA underwentethanol precipitation, and the precipitate was dissolved in 0.1×TE.A260/280 was measured and the DNA concentration was calculated. Thetransfer fragment obtained when pTM6 (FIG. 67) described in ComparativeExample 4-3 was used as a template was 3264 bp, and resulted in asequence within Thraustochytrium aureum Δ4 desaturase gene upstream—SV40terminator sequence—blasticidin resistance gene sequence—ubiquitinpromoter—Thraustochytrium aureum Δ4 desaturase gene (SEQ ID NO: 187).The transfer fragment obtained when pTM8 (FIG. 67) described inComparative Example 4-3 was used as a template was 3935 bp, and resultedin a sequence within Thraustochytrium aureum Δ4 desaturase geneupstream—SV40 terminator sequence—zeocin resistance genesequence—enhanced GFP gene sequence—ubiquitin promoter—Thraustochytriumaureum Δ4 desaturase gene (SEQ ID NO: 188).

The PUFA-PKS pathway associated gene: OrfA gene disruption straindescribed in Comparative Example 2 was cultured for 4 days in a GYculture medium, and cells in the logarithmic growth phase were used ingene transfer. To cells corresponding to OD600=1 to 1.5, 0.625 μg of DNAfragment was transformed by the gene gun method (microcarrier: 0.6micron gold particles, target distance: 6 cm, chamber vacuum: 26 mmHg,rupture disk: 1100 psi). After a recovery time of 4 to 6 hr, thetransgenic cells were spread on a PDA agar plate culture medium(containing 20 mg/mL of zeocin or containing 0.2 mg/mL of blasticidin).As a result, from 100 to 200 cells of drug resistant strain per shotwere obtained.

[Comparative Example 4-5]: Identification of Δ4 Desaturase GeneTargeting Homologous Recombinant

After genome DNA was extracted from the Δ4 desaturase gene disruptionstrain in the Thraustochytrium aureum OrfA disruption strain andThraustochytrium aureum by the method described in Example 2-2,A260/A280 was measured and the DNA concentration was calculated. Usingthe genome DNA as templates. PCR for genome structure confirmation wasperformed using Mighty Amp DNA Polymerase (available from Takara BioInc.). The positions of the primers used, the combinations used inamplification, and the expected sizes of the amplification products areillustrated in FIG. 68. TMO1 was set upstream of the Δ4 desaturase gene,TMO2 was set downstream of the Δ4 desaturase gene, RHO198 (SEQ ID NO:191) and RHO49 (described in Comparative Example 3-3, SEQ ID NO: 153)were set on the blasticidin resistance gene, RHO128 was set on theenhanced GFP gene, and RHO64 (described in Comparative Example 3-4. SEQID NO: 165) was set on the zeocin resistance gene. [TMO1: 23mer: 5′-AAAAGA ACA AGC CCT CTC CTG GA-3′ (SEQ ID NO: 189), TMO2: 23mer: 5′-GAG GTTTGT ATG TTC GGC GGT TT-3′ (SEQ ID NO: 190). RHO198: 26mer: 5′-TGG GGGACC TTG TGC AGA ACT CGT GG-3′ (SEQ ID NO: 191), RHO128: 22mer: 5′-GACCTA CGG CGT GCA GTG CTT C-3′ (SEQ ID NO: 192)]. [PCR cycles: 98° C. 2min/98° C. 10 sec. 68° C. 4 min 30 sec, 30 cycles/68° C. 4 min]. A Δ4desaturase gene disruption strain was obtained, wherein there was noamplification in the wild-type allele (Wt allele), and there wasamplification in the blasticidin resistance gene allele (BlaR allele)and the zeocin resistance gene allele (ZeoR allele) (FIG. 69). Theexperiment reveals that the Thraustochytrium aureum ATCC 34304 straindoes not require nutrients even when the PKS pathway associated gene:OrfA and the Δ4 desaturase gene are deleted.

[Comparative Example 4-6]: Change in Fatty Acid Composition by Δ4Desaturase Gene Disruption in Thraustochytrium aureum OrfA DisruptionStrain

Thraustochytrium aureum ATCC 34304 and the gene disruption strain werecultured according to the method described in Example 2-9, and afterfreeze drying, the fatty acids were methyl-esterified and analyzed usingGC. In GC analysis, measurement was performed using a gas chromatographGC-2014 (available from Shimadzu Corporation) under the followingconditions. Column: HR-SS-10 (30 m×0.25 mm; available from ShinwaChemical Industries Ltd.); column temperature: 150° C.→(5° C./min)→220°C. (10 min); carrier gas: He (1.3 mL/min).

The changes in the fatty acid composition are shown in FIG. 70.Furthermore, FIG. 71 shows the proportion when the wild-type strain istaken as 100%. FIG. 71 shows that, of the total fatty acid composition,ARA is 6.35%, DGLA is 0.90%, ETA is 0.28%, EPA is 6.22%, n-6 DPA is0.21%, and DHA is 0.51%. FIG. 71 shows that, by GC area, LA/DHA is 8.76,GLA/DHA is 1.59, DGLA/DHA is 1.76, ARA/DHA is 12.45. EPA/DHA is 12.20,LA/EPA is 0.72, GLA/EPA is 0.13, DTA/EPA is 1.77, DTA/ARA is 1.73,DTA/DGLA is 12.23, LA/n-6 DPA is 21.29, GLA/n-6 DPA is 3.86, DGLA/n-6DPA is 4.29, ARA/n-6 DPA is 30.24, EPA/n-6 DPA is 29.62, DGLA/LA is0.20, ARA/LA is 1.42, EPA/LA is 1.39, DTA/LA is 2.46, DGLA/GLA is 1.11,ARA/GLA is 7.84, n-6 DPA/DTA is 0.02, DHA/n-3 DPA is 0.03, C20 PUFA/C22PUFA is 0.50, and n-6 PUFA/n-3 PUFA is 0.81.

As a result, when the Δ4 desaturase gene is disrupted in theThraustochytrium aureum OrfA disruption strain, C22:5n-6 (DPA) andC22:6n-3 (DHA) are not substantially biosynthesized, and the Δ4desaturase substrates C22:4n-6 (DTA) and C22:5n-3 (DPA) are accumulated.

Thus, it was demonstrated that in order to create a strain that cannotsubstantially biosynthesize DHA and n-6 DPA from Thraustochytrium aureumATCC 34304, which has both an endogenous elongase-desaturase pathway andan endogenous PUFA-PKS pathway, both a gene of an enzyme constitutingthe elongase-desaturase pathway (for example, the Δ4 desaturase gene)and a PUFA-PKS pathway associated gene needs to be disrupted.

By using the microbial oil obtained in this manner, it is possible toobtain microbial oil having a fatty acid composition in which thecomposition ratios of PUFAs other than DHA and n-6 DPA are increased. Itis possible to produce any PUFAs by modifying the genes of amicroorganism that produces a large amount of DHA. Furthermore, byproducing microbial oil that contains particularly little DHA and n-6DPA, it is possible to produce microbial oil that requires littlerefinement. Additionally, by transforming elongase and desaturase into amicroorganism in this manner, it is possible to obtain a microorganismthat produces microbial oil.

INDUSTRIAL APPLICABILITY

A new “pattern” of biosynthesis pathway of polyunsaturated fatty acids(PUFAs) was discovered in microorganisms called labyrinthulids. Becauseit is possible to provide labyrinthulea that produce PUFAs via only theelongase-desaturase pathway, it is anticipated that PUFAs will be massproduced using only the elongase-desaturase pathway.

1-39. (canceled)
 40. Microbial oil that satisfies not less than onecondition selected from the group consisting of (a) to (g), themicroorganism being a labyrinthulid: (a) ARA is not less than 5% of atotal fatty acid composition; (b) DGLA is not less than 2.5% of thetotal fatty acid composition; (c) ETA is not less than 0.35% of thetotal fatty acid composition; (d) EPA is not less than 4% of the totalfatty acid composition; (e) n-6 DPA is not greater than 0.20% of thetotal fatty acid composition; (f) DHA is not greater than 0.50% of thetotal fatty acid composition; (g) The total of DHA and n-6 DPA is notgreater than 0.7% of the total fatty acid composition.
 41. The microbialoil according to claim 40, wherein the microbial oil satisfies acondition that a value of n-6 DPA/DTA by GC area is not greater than1.5.
 42. The microbial oil according to claim 40, wherein the microbialoil satisfies a condition that a value of DHA/n-3 DPA by GC area is notgreater than
 4. 43. The microbial oil according to claim 40, wherein themicrobial oil satisfies a condition that a value of C20 PUFA/C22 PUFA byGC area is not less than 0.5 and not greater than
 50. 44. The microbialoil according to claim 40, wherein the microbial oil satisfies acondition that a value of n-6 PUFA/n-3 PUFA by GC area is not less than1.8.
 45. The microbial oil according to claim 40, wherein thelabyrinthulid is obtained from a labyrinthulid selected from the groupconsisting of (A) and (B): (A) A labyrinthulid in which a fatty acidcomposition is modified by disruption and/or gene silencing; (B) Alabyrinthulid in which a fatty acid composition is modified bytransforming a gene in addition to disruption and/or gene silencing. 46.A microbial oil obtained from a labyrinthulid genetically modified suchthat a fatty acid composition is modified.
 47. The microbial oilaccording to claim 46, wherein the disrupted and/or silenced gene is aPKS gene, a fatty acid elongase gene, and/or a fatty acid desaturasegene.
 48. The microbial oil according to claim 46, wherein thetransformed gene is a fatty acid elongase gene and/or a fatty aciddesaturase gene.
 49. The microbial oil according to claim 47, whereinthe fatty acid elongase gene is a C20 elongase gene.
 50. The microbialoil according to claim 47, wherein the fatty acid desaturase gene is aΔ4 desaturase gene and/or an ω3 desaturase gene.
 51. The microbial oilaccording to claim 46, wherein a method for disrupting or transforming agene of a labyrinthulid is electroporation, a gene gun method, orgeneediting.
 52. The microbial oil according to claim 46, wherein a methodfor gene silencing of a labyrinthulid is an antisense method or RNAinterference.
 53. A microbial oil obtained from a labyrinthulid selectedfrom the group consisting of (C) and (D): (C) A labyrinthulid havingvery weak or no activity of producing PUFAs via the PUFA-PKS pathway;(D) A labyrinthulid in which the host PUFA-PKS gene is disrupted orsilenced to a very weak level.
 54. The microbial oil according to claim53, wherein the labyrinthulid having very weak or no activity ofproducing PUFAs via the PUFA-PKS pathway is a labyrinthulid belonging tothe genus Parietichytrium or genus Schizochytrium.
 55. The microbial oilaccording to claim 54, wherein the labyrinthulid belonging to the genusParietichytrium is a labyrinthulid belonging to Parietichytriumsarkarianum.
 56. The microbial oil according to claim 54, wherein thelabyrinthulid belonging to the genus Schizochytrium is a labyrinthulidbelonging to Schizochytrium aggregatum.
 57. The microbial oil accordingto claim 55, wherein the microorganism belonging to Parietichytriumsarkarianum is Parietichytrium sp. SEK358 (FERM BP-11405),Parietichytrium sarkarianum SEK364 (FERM BP-11298), or Parietichytriumsp. SEK517 (FERM BP-11406).
 58. The microbial oil according to claim 56,wherein the microorganism belonging to Schizochytrium aggregatum isSchizochytrium aggregatum ATCC
 28209. 59. The microbial oil according toclaim 53, wherein the labyrinthulid in which the host PUFA-PKS gene isdisrupted or silenced to a very weak level belongs to the genusThraustochytrium.
 60. The microbial oil according to claim 59, whereinthe labyrinthulid belonging to the genus Thraustochytrium isThraustochytrium aureum.
 61. The microbial oil according to claim 40,wherein the microbial oil satisfies at least one condition selected fromthe group consisting of (E) to (H): (E) A GC area ratio of ARA aftermodification is not less than 3 times greater than before modification;(F) A GC area ratio of DGLA after modification is not less than 4 timesgreater than before modification; (G) A GC area ratio of ETA aftermodification is not less than 7 times greater than before modification;(H) A GC area ratio of EPA after modification is not less than 7 timesgreater than before modification.
 62. A method for producing themicrobial oil described in claim 61, wherein the microbial oil has avalue of n-6 DPA/DTA by GC area of not greater than 1.5.
 63. The methodfor producing the microbial oil described in claim 61, wherein themicrobial oil has a value of DHA/n-3 DPA by GC area of not greater than4.
 64. The method for producing microbial oil according to claim 61,wherein the microbial oil satisfies a condition that a value of C20PUFA/C22 PUFA by GC area is not less than 0.5 and not greater than 50.65. The method for producing microbial oil according to claim 61,wherein the microbial oil satisfies a condition that a value of n-6PUFA/n-3 PUFA by GC area is not less than 1.8.
 66. A method forproducing microbial oil obtained from a labyrinthulid geneticallymodified such that a fatty acid composition is modified.
 67. The methodfor producing microbial oil according to claim 66, wherein microbial oilis produced in a labyrinthulid genetically modified such that a fattyacid composition is modified, the labyrinthulid being selected from thegroup consisting of (A) and (B): (A) A labyrinthulid in which a fattyacid composition is modified by disruption and/or gene silencing genesilencing; (B) A labyrinthulid in which a fatty acid composition ismodified by transforming a gene in addition to disruption and/or genesilencing.
 68. The method for producing microbial oil according to claim67, wherein the disrupted and/or silenced gene is a PKS gene, a fattyacid elongase gene, and/or a fatty acid desaturase gene.
 69. The methodfor producing microbial oil according to claim 67, wherein thetransformed gene is a fatty acid elongase gene and/or a fatty aciddesaturase gene.
 70. The method for producing microbial oil according toclaim 68, wherein the fatty acid elongase gene is a C20 elongase gene.71. The method for producing microbial oil according to claim 68,wherein the fatty acid desaturase gene is a Δ4 desaturase gene and/or anω3 desaturase gene.
 72. The method for producing microbial oil accordingto claim 67, wherein a method for disrupting or transforming a gene of alabyrinthulid is electroporation, a gene gun method, or gene editing.73. The method for producing microbial oil according to claim 67,wherein a method for gene silencing of a labyrinthulid is an antisensemethod or RNA interference.
 74. The method for producing microbial oilaccording to claim 67, wherein microbial oil is caused to be produced ina labyrinthulid selected from the group consisting of (C) and (D): (C) Alabyrinthulid having very weak or no activity of producing PUFAs via thePUFA-PKS pathway; (D) A labyrinthulid in which the host PUFA-PKS gene isdisrupted or silenced to a very weak level.
 75. A method for producingmicrobial oil that satisfies at least one condition selected from thegroup consisting of (E) to (H): (E) A GC area ratio of ARA aftermodification is not less than 3 times greater than before modification;(F) A GC area ratio of DGLA after modification is not less than 4 timesgreater than before modification; (G) A GC area ratio of ETA aftermodification is not less than 7 times greater than before modification;(H) A GC area ratio of EPA after modification is not less than 7 timesgreater than before modification.
 76. A food, animal feed, medication,or industrial product comprising the microbial oil described in claim 40as a lipid composition.
 77. A labyrinthulid that is genetically modifiedsuch that a produced fatty acid composition is modified and thatproduces the microbial oil described in claim
 40. 78. A method forcreating the labyrinthulid genetically modified such that a producedfatty acid composition is modified described in claim 77.